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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 7 :

G01N 27/327, 33/543, C12Q 1/00, 1/68,
G06F 19/00

(11) International Publication Number: WO 00/45160

(43) International Publication Date: 3 August 2000 (03.08.00)

(21) International Application Number: PCT/US99/17508

(22) International Filing Date: 2 August 1999 (02.08.99)

(30) Priority Data:

PCT/US99/02147 1 February 1999 (01.02.99) US

(63) Related by Continuation (CON) or Continuation-in-Part
(CIP) to Earlier Application

US PCT/US99/02147 (CIP)

Filed on 1 February 1999 (01.02.99)

(71) Applicant (for all designated States except US): SIGNA-

TURE BIOSCIENCE INC. [US/US]; 1450 Rollins Road,
Burlingame, CA 94010 (US).

(72) Inventor; and

(75) Inventor/Applicant (for OS only): HEFTI, John [US/US]; 226
28th Street, San Francisco, CA 94131 (US).

(74) Agents: PERRY, Clifford, B. et al.; Townsend and Townsend
and Crew LLP, 8th floor, Two Embarcadero Center, San
Francisco, CA 941 1 1-3834 (US).

(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB,
GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG,
KP, ICR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MX,
MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA,
ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ,
UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD,
RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK,
ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR,
NE, SN, TD, TG).

Published

With international search report.

(54) Title: METHOD AND APPARATUS FOR DETECTING MOLECULAR BINDING EVENTS
(57) Abstract

Systems and methods are presented for detecting molecular binding events and other environmental effects using the unique dielectric
properties of the bound molecular structure or structures. A molecular binding region is coupled along the surface of a signal path. A
test signal is propagated along the signal path, whereby the test signal couples to the molecular binding region, and in response, exhibits a
signal response.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

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Finland

Lithuania

Austria

France

Luxembourg

Australia

Gabon

Latvia

Azerbaijan

United Kingdom

Monaco

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Georgia

Republic of Moldova

Barbados

Ghana

Madagascar

Belgium

Guinea

The former Yugoslav

Burkina Faso

Greece

Republic of Macedonia

Bulgaria

Hungary

Mali

Benin

Ireland

Mongolia

Brazil

Israel

Mauritania

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Italy

Mexico

Central African Republic

Japan

Niger

Congo

Kenya

Netherlands

Switzerland

Kyrgyzstan

Norway

zvv

Cote d'lvoire

Democratic People's

New Zealand

Cameroon

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Poland

China

Republic of Korea

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Romania

Czech Republic

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Germany

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Ukraine

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United States of America

Uzbekistan

Viet Nam

Yugoslavia

Zimbabwe

WO 00/45160 PCT/US99/17508

METHOD AND APPARATUS FOR DETECTING MOLECULAR

BINDING EVENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of PCT application
No. PCT/US99/02147, entitled "Method and Apparatus for Detecting Molecular Binding
Events," filed February 1, 1999.

BACKGROUND OF THE INVENTION

Virtually every area of biomedical sciences is in need of a system to assay
chemical and biochemical reactions and determine the presence and quantity of particular
analytes. This need ranges from the basic science research lab, where biochemical
pathways are being mapped out and their functions correlated to disease processes, to
clinical diagnostics, where patients are routinely monitored for levels of clinically
relevant analytes. Other areas include pharmaceutical research, military applications,
veterinary, food, and environmental applications. In all of these cases, the presence and
quantity of a specific analyte or group of analytes, needs to be determined.

For analysis in the fields of chemistry, biochemistry, biotechnology,
molecular biology and numerous others, it is often useful to detect the presence of one or
more molecular structures and measure binding between structures. The molecular
structures of interest typically include, but are not limited to, cells, antibodies, antigens,
metabolites, proteins, drugs, small molecules, proteins, enzymes, nucleic acids, and other
ligands and analytes. In medicine, for example, it is very useful to determine the existence
of a cellular constituents such as receptors or cytokines, or antibodies and antigens which
serve as markers for various disease processes, which exists naturally in physiological
fluids or which has been introduced into the system. Additionally, DNA and RNA
analysis is very useful in diagnostics, genetic testing and research, agriculture, and
pharmaceutical development. Because of the rapidly advancing state of molecular cell
biology and understanding of normal and diseased systems, there exists an increasing

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need for methods of detection, which do not require labels such as fluorophores or
radioisotopes, are quantitative and qualitative, specific to the molecule of interest, highly
sensitive and relatively simple to implement.

Numerous methodologies have been developed over the years to meet the
5 demands of these fields, such as Enzyme-Linked Immunosorbent Assays (ELISA),
Radio-Immunoassays (RIA), numerous fluorescence assays, mass spectroscopy,
colorimetric assays, gel electrophoresis, as well as a host of more specialized assays.
Most of these assay techniques require specialized preparations, especially attaching a
label or greatly purifying and amplifying the sample to be tested. To detect a binding

10 event between a ligand and an antiligand, a detectable signal is required which relates to
the existence or extension of binding. Usually the signal is provided by a label that is
conjugated to either the ligand or antiligand of interest. Physical or chemical effects
which produce detectable signals, and for which suitable labels exist, include
radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity

15 to name a few. The label can then be detected by spectrophotometric, radiometric, or
optical tracking methods. Unfortunately, in many cases it is difficult or even impossible
to label one or all of the molecules needed for a particular assay. Also, the presence of a
label may make the molecular recognition between two molecules not function for many
reasons including steric effects. In addition, none of these labeling approaches determines

20 the exact nature of the binding event, so for example active site binding to a receptor is
indistinguishable from non-active-site binding such as allosteric binding, and thus no
functional information is obtained via the present detection methodologies. Therefore, a
method to detect binding events that both eliminates the need for the label as well as
yields functional information would greatly improve upon the above mentioned

25 approaches.

Other approaches for studying biochemical systems have used various
types of dielectric measurements to characterize certain classes of biological systems such
as tissue samples and cellular systems. In the 1950's, experiments were conducted to
measure the dielectric properties of biological tissues using standard techniques for the
30 measurement of dielectric properties of materials known at the time. Since then various
approaches to carrying out these measurements have included frequency domain
measurements, and time domain techniques such as Time Domain Dielectric
Spectroscopy. In these approaches, the experiments were commonly carried out using

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various types of coaxial transmission lines, or other transmission lines and structures of
typical use in dielectric characterization of materials. This included studies to look at the
use and relevance of the dielectric properties of a broad range of biological systems: The
interest has ranged from whole tissue samples taken from various organs of mammalian
5 species, to cellular and 'sub-cellular systems including cell membrane and organelle
effects. Most recently, there have been attempts to miniaturize the above-mentioned
techniques (see e.g., U.S. Patent Nos. 5,653,939; 5,627,322 and 5,846,708) for improved
detection of changes in the dielectric properties of molecular systems. Typically these use
the biological sample — be it tissues, cellular systems, or molecular systems — as a shunt
10 or series element in the electrical circuit topology. This configuration has several
drawbacks, including some substantial limitations on the frequencies useable in the
detection strategy, and a profound limitation on the sensitivity of detecting molecular
systems.

In general, limitations exist in the areas of specificity and sensitivity of

1 5 most assay systems. Cellular debris and non-specific binding often cause the assay to be
noisy, and make it difficult or impossible to extract useful information. As mentioned
above, some systems are too complicated to allow the attachment of labels to all analytes
of interest, or to allow an accurate optical measurement to be performed. Further, a
mentioned above, most of these detection technologies yield no information on the

20 functional nature of the binding event. Therefore, a practical and economical universal
enabling which can directly monitor without a label, in real time, the presence of analytes
or the extent, function and type of binding events that are actually taking place in a given
system would represent a significant breakthrough.

More specifically, the biomedical industry needs an improved general

25 platform technology which has very broad applicability to a variety of water-based or
other fluid-based physiological systems, such as nucleic acid binding, protein-protein
interactions, small molecule binding, as well as other compounds of interest. Ideally, the
assay should not require highly specific probes, such as specific antibodies and exactly
complementary nucleic acid probes; it should be able to work in native environments

30 such as whole blood, cytosolic mixtures, as well as other naturally occurring systems; it
should operate by measuring the native properties of the molecules, and not require
additional labels or tracers to actually monitor the binding event; for some uses it should
be able to provide certain desired information on the nature of the binding event, such as

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whether or not a given compound acts as an agonist or an antagonist on a particular drug
receptor, and not function simply as a marker to indicate whether or not the binding event
has taken place. For many applications, it should be highly miniaturizable and highly
parallel, so that complex biochemical pathways can be mapped out, or extremely small
5 and numerous quantities of combinatorial compounds can be used in drug screening
protocols. In many applications, it should further be able to monitor in real time a
complex series of reactions, so that accurate kinetics and affinity information can be
obtained almost immediately. Perhaps most importantly, for most commercial
applications it should be inexpensive and easy to use, with few sample preparation steps,
10 affordable electronics and disposable components, such as surface chips for bioassays that
can be used for an assay and then thrown away, and be highly adaptable to a wide range
of assay applications.

It is important to note that other industries have similar requirements for
detection, identification or additional analysis. While most applications involve the use
15 of biological molecules, virtually any molecule can be detected if a specific binding

partner is available or if the molecule itself can attach to the surface as described below.

The present invention fulfills many of the needs discussed above and other

needs as well.

20 SUMMARY OF THE INVENTION

The present invention provides systems and methods for detecting molecular
binding events and other environmental effects using the unique dielectric properties of
the bound molecular structure or structures, and the local environment, and also
identifying the presence and concentrations of molecular species, as well as physical
25 properties of the local environment, in a particular biological system.

In a first embodiment of the invention, a method for detecting a molecular
binding event includes the steps of providing a signal path and a molecular binding
region, which is formed along the signal path. A test signal is propagated along the signal
path and couples to the molecular binding region. In response to the coupling, the signal
30 exhibits a response which is indicative of both the molecular binding event and the
molecular binding region itself.

In a second embodiment of the invention, a method for determining the
classification of an unknown ligand is presented. The method comprises the steps of

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providing a signal path coupled to a first molecular binding region having N respective
antiligands for binding to N respective ligand sub-structures. Next a solution containing a
number of unknown ligands is applied to the said molecular binding region. In response,
a second molecular binding region is formed along the signal path, the second molecular
5 binding region having N ligands. N respective test signals are propagated to the N

respective ligands. N known signal responses defining a known ligand classification are
provided. Finally, each of the test signals couples to the N ligand/antiligand complexes,
and in response exhibits N respective measured responses indicative of the presence of
each of said N sub-structures, so that if a predetermined number of said N known signal

10 responses correlates within a predefined range with the N measured responses, the ligand
is determined to be within the known classification.

In a third embodiment of the invention, a method for identifying an
unknown molecular binding event is presented. The method includes the steps of
providing a signal path, applying a first solution containing a first ligand over the signal

15 path, and forming, in response, a first molecular binding region along the signal path,
whereby the first molecular region includes the first ligand and is positioned along the
signal path and the first solution. A first test signal is propagated along the signal path,
the portion of which includes the molecular binding region comprises a continuous
transmission line, whereby the signal couples to the molecular binding region and in

20 response exhibits a first signal response. A known signal response corresponding to a

known molecular binding event is provided and the first signal response is then compared
to the known signal response, wherein if the first signal response correlates to the known
signal response within a predefined range, the unknown molecular binding event
comprises the known molecular binding event.

25 In a fourth embodiment of the invention, a method for quantitating an

unknown concentration of ligands in solution is presented. The method includes the steps
of providing a signal path which is coupled to a first molecular binding region having at
least one antiligand, applying a solution having a known concentration of ligands over the
molecular binding region, and propagating a test signal along the signal path. Next a first

30 signal response is measured and an extrapolation algorithm is generated. A second test
signal is subsequently propagated and a second signal response is measured. The second
signal response is then correlated to the algorithm.

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In a fifth embodiment of the invention, a bio-electrical interface is
provided for detecting the presence of a ligand in a solution. The bio-electrical interface
includes a signal path, a solution for providing the ligand and a molecular binding region.
The molecular binding region includes the ligand and is coupled along the signal path and
5 the solution.

In a sixth embodiment of the invention, a bio-assay device is provided for
detecting one or more properties associated with a molecular binding region, such as the
presence of a ligand, using a test signal. The apparatus includes a signal path having a
first port and a second port for communicating the test signal, and a continuous

10 conductive region therebetween. The bio-assay device further includes a molecular
binding region, which may have a ligand, and which is coupled to the signal path. The
bio-assay device may further include a solution coupled to said molecular binding region,
which may transport the ligand to the molecular binding region.

In a seventh embodiment of the invention, a system for detecting a

1 5 molecular binding event is presented. The system includes a signal source for launching
a test signal, a bio-assay device coupled to said signal source and a second detector
coupled to the bio-assay device. The bio-assay device includes a signal path and a first
molecular binding region, which may include a ligand or antiligand, and which may be
coupled to a solution and the signal path. The test signal propagates along the signal path,

20 which is continuous throughout the region of the molecular binding region, and couples to
the molecular binding region, and in response exhibits a signal response which indicates
the presence of said molecular binding event.

In one aspect, the present invention is the use of the interaction of
electromagnetic radiation, typically between about 1 MHz and 1000 GHz, with molecular

25 structures in a molecular binding region to determine properties of the structures, such as
dielectric properties, structural properties, binding events and the like. Also, the present
invention uses a test signal on a bio-electrical interface having a signal path along which
the molecular binding region is coupled to detect analytes therein.

The nature and advantages of the present invention will be better

30 understood with reference to the following drawings and detailed description.

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BRIEF DESCRIPTION OF THE DRAWTNGS
Fig. 1 A illustrates one embodiment of the bio-assay system in accordance
with the present invention.

Fig. IB illustrates a second embodiment of the bio-assay system in
5 accordance with the present invention.

Fig. 1 C illustrates a cross-section view of the bio-assay system shown in

Fig. IB.

Fig. ID illustrates one embodiment of a molecular binding region in
accordance with the present invention.
10 Fig. IE illustrates one embodiment of a molecular binding region having

multiple antiligands which are spatially separated in accordance with the present
invention.

Fig. IF illustrates one embodiment of a molecular binding region having
multiple classes of anitligands in accordance with the present invention.
1 5 Fig. 1 G illustrates a molecular binding region comprising one or more

cells in accordance with the present invention.

Fig. 1H illustrates a molecular binding region comprising cell membranes
and membrane associated structures in accordance with the present invention.

Fig. 2 A illustrates one embodiment of the bio-assay device in accordance
20 with the present invention.

Fig. 2B illustrates a second embodiment of the bio-assay device in
accordance with the present invention.

Fig. 3 illustrates one embodiment of the binding surface chemistry which
occurs along the conductive layer of the bio-electrical interface.
25 Fig. 4A illustrates one embodiment of an equivalent circuit model for the

bio-electrical interface structure shown in Fig. 2A.

Fig. 4B illustrates one embodiment of a circuit corresponding to the
equivalent circuit model shown in Fig. 4A.

Fig. 4C illustrates one embodiment of an equivalent circuit model for the
30 bio-electrical interface structure shown in Fig. 2B.

Fig. 4D illustrates one embodiment of a circuit corresponding to the
equivalent circuit model shown in Fig. 4C.

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Figs. 5A-5G illustrate specific embodiments of the bio-electrical interface
implemented in a two conductor circuit topology in accordance with the present
invention.

Fig. 6 A illustrates one embodiment of a method for detecting molecular
5 binding events in accordance with the present invention.

Fig. 6B illustrates one embodiment of a method for detecting secondary
and higher-order binding events in accordance with the present invention.

Fig. 6C illustrates one embodiment of a method for measuring dielectric

changes of the molecular binding region in accordance with the present invention..
10 Fig. 6D illustrates one embodiment of a method for identifying a ligand in

an unknown solution in accordance with the present invention..

Fig. 6E illustrates one embodiment of a method for identifying the class of
a ligand in accordance with the present invention.

Fig. 6F illustrates one embodiment of a method for quantitating the ligand
1 5 concentration of a solution in accordance with the present invention.

Fig. 6G illustrates one embodiment of a method for providing a self-
diagnostic capability of the bio-assay device in accordance with the present invention.

Fig. 7A illustrates one embodiment of a computer system for executing a
software program designed to perform each of the methods shown in Figs. 6A-G.
20 Fig. 7B illustrates a simplified system block diagram of a typical computer

system used to execute a software program incorporating the described method.

Fig. 8 A illustrates one embodiment of a frequency measurement system in
accordance with the present invention.

Fig. 8B illustrates a first frequency response measured which can be used
25 to detect or identify a molecular structure in accordance with the present invention.

Fig. 8C illustrates a second frequency response which can be used to
detect or identify a molecular structure in accordance with the present invention.

Fig. 9 illustrates a second embodiment of a frequency measurement system
in accordance with the present invention.
30 Fig. 10 illustrates one embodiment of a time domain measurement system

in accordance with the present invention.

Fig. 1 1 illustrates one embodiment of a dielectric relaxation measurement
system in accordance with the present invention.

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Figs. 12A-B illustrate the return loss and transmission loss measurements,
respectively, of the primary binding of urease to an ITO surface.

Figs. 12C and 12D illustrate the transmission loss measurements of the
primary binding effects of collagenase and lysozyme.

Fig. 12E illustrates the transmission loss response of bound and unbound

dextran.

Fig. 12F illustrates the response of con- A unbound and bound to glucose.
Fig.l2G illustrates the transmission loss of biotin/Avidin relative to the
Avidin response.

Fig. 12H illustrates the results of a competition titration between dextran

and glucose.

Fig. 121 illustrates the return loss of con-A as a function of glucose
concentration at resonance.

Fig. 12J illustrates the transmission loss of DNA/Polylysine complexes
relative to the Polylysine response.

Fig. 1 2K illustrates the change in the transmission loss response as a
function of pH for a series of buffers at 1 00 MHz, 1 GHz, and 1 0 GHz.

Fig. 1 2L illustrates the change in the transmission loss response as a
function of ionic concentration for a series of buffers at 100 MHz, 1 GHz, and 10 GHz.

Fig. 12M illustrates the transmission loss response for 10 samples of whole
blood probed at 1 GHz indicating detection capability in a complex environment.

Fig. 1 2N illustrates the result of avidin binding indicating quadrapole
moment detection.

DESCRIPTION OF THE SPF.PTFT C EMBODTMKNTS
Table of Content*

I. Definition of Terms

II. Introduction

A. Bio-Assay System

B. Chemistry of the System
HI. The Bio-Assay Device

A. Device Structure

B. Binding Surface Chemistry

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C. Bio-Electrical Interface

D. Specific Embodiments

IV. Measurement Methodology

A. General Overview
5 B. Detecting Molecular Binding Events

C. Detecting Changes in the Dielectric Properties

D. Identifying Molecular Binding Events

E. Identifying Classes of Bound Molecular Structures
R Quantitating Concentrations

10 G. Bio- Assay Device Self-Calibration

V. Measurement Systems

A. Frequency Measurement System

B. Time Domain Measurement System

C. Dielectric Relaxation Measurement System
15 VI. Examples

VTL Applications

I. Definition of Terms

As used herein, the terms biological "binding partners" or

20 "ligand/antiligand" or "ligand/antiligand complex" refers to molecules that specifically
recognize {e.g. bind) other molecules to form a binding complex such as antibody-
antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc. Biological
binding partners need not be limited to pairs of single molecules. Thus, for example, a
single ligand may be bound by the coordinated action of two or more "anti-ligands".

25 As used herein, the term "ligand" or "analyte" or "marker" refers to any

molecule being detected. It is detected through its interaction with an antiligand, which
specifically or non-specifically binds the ligand, or by the ligand's characteristic dielectric
properties. The ligand is generally defined as any molecule for which there exists another
molecule (i.e. an antiligand) which specifically or non-specifically binds to said ligand,

30 owing to recognition of some portion of said ligand. The antiligand, for example, can be
an antibody and the ligand a molecule such as an antigen which binds specifically to the
antibody. In the event that the antigen is bound to the surface and the antibody is the
molecule being detected, for the purposes of this document the antibody becomes the

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11 )

ligand and the antigen is the antiligand. The ligand may also consist of cells, cell
membranes, organelles and synthetic analogues thereof.

Suitable ligands for practice of this invention include, but are not limited
to antibodies (forming an antibody/epitope complex), antigens, nucleic acids (e.g. natural
or synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA, etc.), lectins, sugars (e.g.
forming a lectin/sugar complex), glycoproteins, receptors and their cognate ligand (e.g.
growth factors and their associated receptors, cytokines and their associated receptors,
signaling receptors, etc.), small molecules such as drug candidates (either from natural
products or synthetic analogues developed and stored in combinatorial libraries),
metabolites, drugs of abuse and their metabolic by-products, co-factors such as vitamins
and other naturally occurring and synthetic compounds, oxygen and other gases found in
physiologic fluids, cells, cellular constituents cell membranes and associated structures,
other natural products found in plant and animal sources, other partially or completely
synthetic products, and the like.

As used herein, the term "antiligand" refers to a molecule which .
specifically or nonspecifically binds another molecule (i.e., a ligand). The antiligand is
also detected through its interaction with a ligand to which it specifically binds or by its
own characteristic dielectric properties. As used herein, the antiligand is usually
immobilized on the surface, either alone or as a member of a binding pair that is
immobilized on the surface. In some embodiments, the antiligand may consist of the
molecules on the signal path or conductive surface. Alternatively, once an antiligand has
bound to a ligand, the resulting antiligand/ligand complex can be considered an
antiligand for the purposes of subsequent binding.

As used herein, the term "specifically binds" when referring to a protein or
polypeptide, nucleic acid, or receptor or other binding partners described herein, refers to
a binding reaction which is determinative of the cognate ligand of interest in a
heterogenous population of proteins and/or other biologies. Thus, under designated
conditions (e.g. immunoassay conditions in the case of an antibody), the specified ligand
or antibody binds to its particular "target" (e.g. a hormone specifically binds to its
receptor) and does not bind in a significant amount to other proteins present in the sample
or to other proteins to which the ligand or antibody may come in contact in an organism
or in a sample derived from an organism. Similarly, nucleic acids may hybridize to one
another under preselected conditions.

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As used herein, the terms "isolated" "purified" or "biologically pure" refer
to material which is substantially or essentially free from components that normally
accompany it as found in its native state.

As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form, and unless otherwise
limited, encompasses known analogs of natural nucleotides that can function in a similar
manner as naturally occurring nucleotides.

As used herein, the terms "polypeptide", "peptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues. The terms apply to amino
acid polymers in which one or more amino acid residue is an artificial chemical analogue
of a corresponding naturally occurring amino acid, as well as to naturally occurring amino
acid polymers.

As used herein, the term "antibody" refers to a protein consisting of one or
more polypeptides substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad
immunoglobulin variable region genes. Light chains are classified as either kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprise
a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each
pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-
terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids
primarily responsible for antigen recognition. The terms variable light chain (VL) and
variable heavy chain (VH) refer to these light and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of well-
characterized fragments produced by digestion with various peptidases. Thus, for
example, pepsin digests an antibody below the disulfide linkages in the hinge region to
produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab) f 2 may be reduced under mild conditions to break the disulfide
linkage in the hinge region thereby converting the (Fab') 2 dimer into an Fab 1 monomer.
The Fab* monomer is essentially an Fab with part of the hinge region (see, Fundamental
Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of

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other antibody fragments). While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that such Fab 1 fragments may
be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody fragments either produced
5 by the modification of whole antibodies or synthesized de novo using recombinant DNA
methodologies. Preferred antibodies include single chain antibodies, more preferably
single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are
joined together (directly or through a peptide linker) to form a continuous polypeptide.

A single chain Fv ("scFv" or "scFv") polypeptide is a covalently linked

1 0 VH: : VL heterodimer which may be expressed from a nucleic acid including VH- and VL-
encoding sequences either joined directly or joined by a peptide-encoding linker. Huston,
et al. (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883. A number of structures for
converting the naturally aggregated- but chemically separated light and heavy
polypeptide chains from an antibody V region into an scFv molecule which will fold into

15 a three dimensional structure substantially similar to the structure of an antigen-binding
site. See, e.g. U.S. Patent Nos. 5,091,513 and 5,132,405 and 4,956,778.

An "antigen-binding site" or "binding portion" refers to the part of an
immunoglobulin molecule that participates in antigen binding. The antigen binding site is
formed by amino acid residues of the N-terminal variable ("V") regions of the heavy

20 ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the
heavy and light chains are referred to as "hypervariable regions" which are interposed
between more conserved flanking stretches known as "framework regions" or "FRs".
Thus, the term "FR" refers to amino acid sequences that are naturally found between and
adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three

25 hypervariable regions of a light chain and the three hypervariable regions of a heavy
chain are disposed relative to each other in three dimensional space to form an antigen
binding "surface". This surface mediates recognition and binding of the target antigen.
The three hypervariable regions of each of the heavy and light chains are referred to as
"complementarity determining regions" or "CDRs" and are characterized, for example by

30 Kabat et al. Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health
and Human Services, Public Health Services, Bethesda, MD (1987).

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As used herein, the terms "immunological binding" and "immunological
binding properties" refer to the non-covalent interactions of the type which occur between
ail immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
As used herein, a biological sample is a sample of biological tissue or fluid that, in a
5 healthy and/or pathological state, that is to be assayed for the analyte(s) of interest. Such
samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g.,
white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid,
or cells therefrom. Biological samples may also include sections of tissues such as frozen
sections taken for histological purposes. Although the sample is typically taken from a

10 human patient, the assays can be used to detect the analyte(s) of interest in samples from
any mammal, such as dogs, cats, sheep, cattle, and pigs. The sample may be pretreated as
necessary by dilution in an appropriate buffer solution or concentrated, if desired. Any of
a number of standard aqueous buffer solutions, employing one of a variety of buffers,
such as phosphate, Tris, or the like, preferably at physiological pH can be used.

15 As used herein, the term "signal path" refers to a transmission medium

along or through the bio-electrical interface which is capable of supporting a-c time-
varying or a DC static electromagentic field. A non-exhaustive list of signal paths
include conductive and dielectric waveguide structures, multiple dielectric and onductor
transmission mediums such as transverse electromagnetic (TEM) transmission lines,

20 transmission lines with three or more conductive elements which support TE, TM or
TEM mode propagation such as quadrupolar and octupolar lines, coupled waveguides,
resonant cavity structures which may or may not be coupled, other non-modal structures
like wires, printed circuits, and other distributed circuit and lumped impedance
conductive structures, and the like. The signal path may structurally comprise the signal

25 plane, the ground plane, or a combination of both structures. Typically, the signal path is
formed along a direction which is non-orthogonal to the surface of the MBR. In
embodiments in which the signal path consists of a conductive layer or region, the
conductive region extends continuously over that range. In embodiments in which the
signal path is non-metallic, i.e., a dielectric waveguide, the signal path is defined as the

30 path having the least amount of signal loss or as having a conductivity of greater than 3
mhos/m.

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As used herein, the terms "molecular binding region" or "MBR" refers to a
region having of at least one molecular structure (i.e., an analyte, antiligand, or a
ligand/antiligand pair, etc.) coupled to the signal path along the bio-electrical interface.
The molecular binding region may consist of one or more ligands, antiligands,
5 ligand/antiligand complexes, linkers, matrices of polymers and other materials, or other
molecular layers/structures described herein. Further, the molecular binding region may
be extremely diverse and may include one or more components including matrix layers
and/or insulating layers, which may have one or more linking groups. The MBR is
coupled to the signal path either via a direct or indirect physical connection or via

1 0 electromagnetic coupling when the ligand is physically separated from the signal path.
The MBR may be of a derivatized surface such as by thiol linkers biotinylated metals and
the like, all in accordance with standard practice in the art

As used herein, the term 'binding event" refers to an interaction or
association between a minimum of two molecular structures, such as a ligand and an

15 antiligand. The interaction may occur when the two molecular structures as are in direct
or indirect physical contact or when the two structures are physically separated but
electromagnetically coupled therebetween. Examples of binding events of interest in a
medical context include, but are not limited to, ligand/receptor, antigen/antibody,
enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid mismatches,

20 complementary nucleic acids and nucleic acid/proteins. Alternatively, the term "binding
event" may refer to a single molecule or molecular structure described herein, such as a
ligand, or an antiligand/ligand complex, which is bound to the signal path. In this case
the signal path is the second molecular structure.

As used herein, the term "Ligand/antiligand coniplex" refers to the ligand

25 bound to the antiligand. The binding may be specific or non-specific, and the bonds are
typically covalent bonds, hydrogen bonds, immunological binding, Van der Waals forces,
or other types of binding.

As used herein, the term "coupling" refers to the transfer of energy
between two structures either through a direct or indirect physical connection or through

30 any form of signal coupling, such as electrostatic or electro-magnetic coupling.

As used herein, the term "test signal" refers to a signal propagating at any
useful frequency defined within the electromagnetic spectrum. For examples, the test
signal frequency is at or above 1 MHz, such as 5 MHZ 10 MHz, 20 MHz, 45 MHz, 100

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MHz, 500 MHz, 1 GHz, 5 GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz, 500 GHz, 1000
GHz and frequencies ranging therebetween.

As used herein, the term "enzyme," refers to a protein which acts as a
catalyst to reduce the activation energy of a chemical reaction in other compounds or
"substrates", but is not a final product in the reaction.

As used herein, the term "solution" or "sample" includes a material in
which a ligand resides. A non-exhaustive list of solutions includes materials in solid,
liquid or gaseous states. Solid solutions may be comprised of naturally-occurring or
synthetic molecules including carbohydrates, proteins, oligonucleotides, or alternatively,
any organic polymeric material, such as nylon, rayon, dacryon, polypropylene, teflon,
neoprene, delrin or the like. Liquid solutions include those containing an aqueous, organic
or other primary components, gels, gases, and emulsions. Exemplary solutions include
celluloses, dextran derivatives, aqueous solution of d-PBS, Tris buffers, deionized water,
blood, physiological buffer, cerebrospinal fluid, urine, saliva, water, organic solvents. The
solution is used herein to refer to the material in which the ligand and/or antiligand are
applied to the binding surface. The solution contains the sample to be analyzed.

As used herein, the term "linking group" or "linker" refers to chemical
structures which are used to attach any two components on the bio-assay device. The
linking groups thus have a first binding portion that binds to one component, such as the
conductive surface, and have a second binding portion that binds to another component
such as the matrix or the antiligand.

As used herein, the term "bio-assay device" refers to a structure in which
the molecular binding region is formed. The bio-assay device may consist of a surface,
recessed area, or a hermetically sealed enclosure, all of which may be any particular size
or shape.

As used herein, the "bio-assay system" refers to the bio-assay device as
described above, in connection with the components necessary to electromagnetically
probe and detect the bio-assay device. These components include, but are not limited to,
the signal path(s), substrate(s), electronic devices such as signal generators, oscilloscopes,
and vector analyzers necessary to probe to and detect signals from the bio-assay device,
microchips and microprocessors which can probe and detect electromagnetic signals and
analyze data, and the like.

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As used herein, the term "resonant" or "resonance" refers generally to a
rapidly changing dielectric response as a function of frequency.

As used herein, "bio-electrical interface" refers to an interface structure
between a signal path for supporting the propagation of a test signal and a molecular
5 binding region.

As used herein, the term "matrix" or "binding matrix" refers to a layer of
material on the bioassay chip that is used as a spacer or to enhance surface area available
for binding or to optimize orientation of molecules for enhanced binding, or to enhance
any other property of binding so as to optimize the bio-assay device. The matrix layer
1 0 may be comprised or carbohydrates such as dextran, poly amino acids, cross-linked and
non-cross linked proteins, and the like.

II. Introduction

15 A. The Bio- Assay System

The present invention makes use of the observation that a vast number of
molecules can be distinguished based upon the unique dielectric properties most
molecules exhibit. These distinguishing dielectric properties can be observed by coupling
a signal to the bound molecular structure. The unique dielectric properties modulate the

20 signal, giving it a unique signal response. The unique signal response can then be used to
detect and identify the ligands and other molecules which make up the molecular binding
region.

Fig. 1 A illustrates one embodiment of a bio-assay system 1 00 in
accordance with the present invention. The system 100 is illustrated in a two conductor,

25 signal-plane ground-plane, circuit topology which may be realized in a multitude of
architectures including lumped or distributed element circuits in microstrip, stripline,
coplanar waveguide, slotline or coaxial systems. Moreover, those of skill in the art of
electronics will readily appreciate that the system may be easily modified to a single
conductor waveguide system, or a three or more conductor system.

30 As illustrated, the system 100 includes a signal source 110, transmission

lines 120, a ground plane 130, a bio-assay device 150, and a signal detector 160. The
illustrated embodiment shows two transmission lines 120 coupled to the bio-assay device
150, although in alternative embodiments a single transmission line may be coupled to the

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bio-assay device or further alternatively, three or more transmission lines may coupled to
the bio-assay device 150. Transmission lines 120 are formed from a material which can
support the propagation of a signal over the desired frequency of operation. Transmission
lines 120 are realized as a conductive layer, such as gold, deposited on a substrate, such
5 as alumina, diamond, sapphire, polyimide, or glass using conventional photolithography
or semiconductor processing techniques.

The system 1 00 farther includes a bio-assay device 1 50 coupled to the
transmission lines 120. The bio-assay device 150 contains a supporting substrate 151
onto which a conductive layer 153 is disposed. The conductive layer 153 forms an

1 0 interface for supporting the propagation of a test signal. The supporting substrate 1 5 1

may consists of any insulating material such as glass, alumina, diamond, sapphire, silicon,
gallium arsenide or other insulating materials used in semiconductor processing.

A molecular binding region (MBR) 1 56 is coupled to one or more areas of
the signal path 153. As those of skill in the art of electronics will appreciate, coupling

1 5 may occur either through a direct connection between the signal path 1 53 and MBR 156
as illustrated, or alternatively through signal coupling, further described below.

The MBR 1 56 is primarily composed of one or more ligands, although
other molecules and structures may also be included, as described herein. The MBR 156
may consist of only one bound ligand tier, for instance in the case of primary binding, or

20 it may consist of two, three, four, five or more bound ligand tiers, in the instances where
there are secondary or higher-order binding events occurring. Multiple ligand tiers may
occur at different binding surfaces 155 over the same signal path 153.

In the illustrated embodiment, dielectric substrate 158 is located between
solution 157 and ground plane 159. In the illustrated embodiment, dielectric layer 158

25 and ground plane 159 are located within the bio-assay device 150, although in alternative
embodiments, one or both may be located externally. Furthermore, the MBR 156 and
solution 157 arrangement may be switched and moved towards the ground plane
alternatively, or in addition to its proximity to the signal path 153.

The system 100 includes a signal source 110 which launches the test signal

30 onto the transmission line 120 and towards the bio-assay device 1 50. A signal detector
160 is positioned along the transmission path to detect the resulting signal (either
reflected or transmitted or both). When the signal propagates along the signal path 153 of
the bio-assay device 150, the dielectric properties of the MBR 156 modulates the test

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signal. The modulated signal can then be recovered and used to detect and identify the
molecular binding events occurring within the bio-assay device, further described below.
In an alternative embodiment of the invention, detection and identification
of a ligand, antiligand/ligand complex or other molecular structure described herein is
5 possible when it is physically separated from the signal path 153. In this embodiment, the
ligand is separated from but electrically or electromagnetically coupled to the signal path
153. The coupling between the signal path 153 and the suspended ligand will alter the
response of the test signal propagating along the signal path 153, thereby providing a
means for detecting and/or identifying it. The maximum separation between the signal

10 path 153 and suspended ligand is determined by such factors as the effective dielectric
constant of the medium between the signal path 153 and the ligand, the total coupling
area, the sensitivity of the signal detector, concentration of the ligands in solution, and the
desired detection time. Separation distances are typically on the order of 10 -1 m, 10* 2 m 10*
3 m, ICrV 10" 5 m, 10' 6 m, 10" 7 m, 10" 8 m, 10' 9 m, 10" 10 m or range anywhere therebetween.

15 In some embodiment, such as cell based assays, the MBR may be

electromagnetically coupled to the signed path through the solution. Thus, cells, and in
particular cell membranes and membrane-based structures may couple to the signal.

Fig. IB illustrates a second embodiment of the bio-assay system
comprising an array of resonant microstrip circuits 170. Each resonant circuit 170

20 consists of a transmission line 172 terminating in an open-circuited stub 176. Those
skilled in the art of circuit design will appreciate other resonant structures may be
employed in lumped element, distributed, or a combination of both circuit topologies.

Fig. 1C illustrates a cross-section view of one resonant circuit 170. The
open-circuited stub 176 forms the bio-electrical interface of the resonant circuit 170 and

25 closely parallels the bio-electrical interface shown in Fig. 1 A. In particular, the open-
circuited stub 176 consists of an signal path 176a deposited on a dielectric layer 176b, and
is positioned above ground plane 176c.

In this embodiment, the MBR 1 76d is coupled via a direct connection to
transmission line 176a. The MBR 176d can bind along the signal path in a specific or

30 non-specific manner. As above, the subject molecular structure may be suspended from
but electrically coupled or electromagnetically coupled to the signal path 176a to provide
binding event detection and identification information.

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The dimensions of the signal path 176a are influenced by considerations
such as the desired measurement time (a larger area resulting in faster detection time), the
desired resonant frequency f^s, certain impedance matching conditions to achieve higher
efficiency or cause discontinuities to highlight binding events, and the process by which
5 the entire array is formed. For instance, if conventional microwave photolithography is
used, the binding surface area may range from lO'W to 10"W using a relatively thick
dielectric layer such as alumina, diamond, sapphire, duriod or other conventional
substrate materials. Alternatively, if semiconductor processing is used, the binding
surface area may range from 10' 6 m 2 to 10" 12 m 2 using a relatively thin dielectric layer of

10 silicon or gallium arsenide.

Using conventional microwave design techniques or CAD tools such as
Microwave Spice™, EEsof Touchstone™ and Libra™, the length and impedance of the
transmission line 172, the dimensions of the signal path 176a, and the thickness and
dielectric constant of the dielectric layer 176b can be selected such that the resonant

1 5 structure exhibits a resonant signal response at a desired resonant frequency point f res .
The desired resonant frequency f res point is typically the frequency range over which the
molecules of interest exhibit a dramatic change in their dielectric properties, the
measurement of which will enable their detection. Alternatively, the resonant frequency
point f res can be defined as the center of the desired test frequency range to allow for the

20 widest range of signal detection. In the illustrated embodiment, the resonant frequency
f rcs includes 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz, 5 GHz, 10 GHz,
30 GHz, 50 GHz, 100 GHz, 500 GHz, 1,000 GHz or frequencies ranging therebetween.

During measurement, the solution 1 76e is applied over one or more of the
open-circuited stubs 172. A MBR 176d is formed when one or molecules within the

25 solution bind to the signal path 176a. In this instance, the MBR 176d and the solution
electrically behave as a parasitic circuit, further described below, which causes the
resonant frequency point f^s to shift above or below its original resonant frequency point.
This shift in frequency can be detected, and is used to indicate the occurrence of a
molecular binding event. The signal response may also be interrogated over a wide

30 spectrum to ascertain the identity of the bound molecular structure, as described below.
Each resonant circuit 1 70 may be fabricated to bind different molecular structures and
each resonant circuit 170 be made addressable, thereby permitting simultaneous detection

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and identification of a large numbers of molecular structures within the same solution. In
an alternative embodiment, each resonant circuit 170 may be designed to exhibit a distinct
resonant frequency, in which case all of the resonant circuits 170 may be interrogated
over a continuous frequency spectrum to determine molecular binding.
5 The signal path 153 is designed to support the propagation of an

electromagnetic signal at the desired test frequency. Many configurations are possible,
one example being a sputtered gold transmission line operable between D.C. and 110
GHz. In another embodiment, the signal consists of a dielectric medium, such as the
MBR itself. In this embodiment, the signal path blocks DC voltages and currents but

10 otherwise supports the propagation of the desired test signal, occurring at frequencies, for
instance 1 MHz, 5 MHz 10 MHz, 20 MHz, 45 MHz, 80 MHz, 100 MHz, 250 MHz, 500
MHz, 750 MHz, 1 GHz, 2.5 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 18 GHz, 20 GHz,
22 GHz, 24 GHz, 26 GHz, 30 GHz, 33 GHz, 40 GHz, 44 GHz, 50 GHz, 80 GHz, 96
GHz, 100 GHz, 500 GHz, 1000 GHz, or frequencies ranging therebetween. Accordingly,

15 the signal path 153 is designed using high frequency circuit design techniques, known in
the art. Such design techniques include impedance matching the signal path 153 to the
interconnecting structures, minimizing the insertion loss of the signal path 153, and
minimizing the Voltage Standing Wave Ratio (VSWR) of the signal path 153. In the
preferred embodiment of the present invention, the signal path 153 and MBR 156 are

20 oriented in a non-orthogonal orientation.

B. Chemistry of the System

The chemistry of the system generally occurs within the bio-assay device,
and in particular along the conductive layer (signal path in Figs 1 A-1C). The conductive

25 layer is fabricated from materials and having a morphology which is conducive to support
the propagation of the high frequency test signal. The conductive surface is constructed
from materials exhibiting appropriate conductivity over the desired test frequency range
as well as possessing good molecular binding qualities as described above. Such
materials include, but are not limited to gold, indium tin oxide (ITO), copper, silver, zinc,

30 tin, antimony, gallium, cadmium, chromium, manganese, cobalt, iridium, platinum,
mercury, titanium, aluminum, lead, iron, tungsten, nickel, tantalum, rhenium, osmium,
thallium or alloys thereof. The conductive layer may also be formed from
semiconducting materials which may be either crystalline or amorphous in structure,

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including chemically doped or pure carbon, silicon, germanium, gallium-arsenide, idium-
gallium arsenide, or the like. The conductive material may also be formed from polymers
especially those that are conductive such as polyacetylene, polythiophene and the like.
The conductive layer may be thick or only several molecular layers in depth as the
5 application requires. The conductive layer may be comprised of an evaporated thin metal
layer or an epitaxial layer of gallium arsenide or other semiconductor materials rendered
conductive through known semiconductor processing techniques. In addition, the
conductive layer may be derivatized, the process by which is well known, e.g., see Kumar
et al., "Patterned Self-Assembred Monolayer and Mesoscale Phenomena," Accounts of

10 Cemical Research. 28:219-226 (1995).

The conductive layer is additionally fabricated from materials and having a
morphology which is conducive to facilitate molecular binding, Ligands may bind
directly, indirectly through other molecular structures, or through both configurations to
bind to the conductive layer. The range of molecules that may bind to the conductive

15 layer include but are not limited to proteins, nucleic acids, small molecules, saccharides,
lipids, and any other molecule of interest. The chemistry may involve only a single
species of molecules attached to the surface, a whole array of different species attached to
the surface, or multiple binding events between species directly attached to the surface
and ligands of interest in the solution,

20 The typical chemistry involved in attaching a ligand to the conductive

layer will in general depend on the nature of the ligand and any antiligand to which it
binds, and their functions in the assay. A list of possible types of interactions that may
occur on the surface include but are not limited to: Protein/protein interactions,
DNA/protein interactions, RNA/protein interactions, nucleic acid hybridization, including

25 base pair mismatch analysis, RNA/RNA interactions, tRNA interactions,

Enzyme/substrate systems, antigen/antibody interactions, small molecule/protein
interactions, drug/receptor interactions, membrane/receptor interactions, conformational
changes in solid phase ligands, protein/saccharide interactions, and lipid/protein
interactions.

30 The actual surface chemistry may be described in one embodiment as

primary binding and secondary binding. Additional regions of molecular binding may
also occur. Primary binding refers to the attachment of an antiligand to the conductive
surface, which can be done through the assistance of a linker molecule. Secondary

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binding refers to the binding of a ligand to the antiligand, which may be another molecule
in the MBR or directly to the conductive surface itself. Typically, the binding involves a
liquid phase ligand binding to an immobilized solid phase antiligand. For example,
primary binding could be the attachment of a specific antibody to the conductive layer of
5 . the bioassay device and secondary binding would involve the binding of a specific

antigen in a sample solution to the antibody. Alternatively, secondary binding may be the
direct attachment of a protein to the conductive surface, such as the amine terminus of a
protein attaching directly to a gold conductive layer.

The aforementioned binding results in the formation of a molecular

1 0 binding region (MBR) 1 80 along one or more areas of the conductive layer, one
embodiment of which is illustrated in Fig. ID. In this embodiment, the MBR 180
optionally consists of a first linker 181, an insulator 1 82, a second linker 183, a matrix
184, a third linker 185, an antiligand layer 186, and a ligand layer 187.

First linker 181 provides attachment between insulating layer 182 and

15 conductive layer (not shown). First linker 181 consists of molecule such as thiols, amines,
amides, or metals such as chromium or titanium. Insulating layer 1 82 provides a barrier
between the conductive layer and the MBR 180 and solution (not shown). Insulating
layer 182 may provide a hermetic barrier to prevent structural deterioration of conductive
layer due to exposure to the MBR and/or solution. Alternatively, or in addition,

20 insulating layer 182 may consist of an electrically non-conductive material to prevent the
flow of DC or low frequency energy from the conductive layer to the MBR and/or
solution which could interfere with the measurement. The insulating layer may include
polyimide, alumina, diamond, sapphire, non-conductive polymers, semiconductor
insulating material such as silicon dioxide or gallium arsenide or other materials which

25 provide hermetic and/or electrically insulating characteristics. The insulating layer may
also consist of air, or another gaseous substance, in which case linker 181 may be deleted.

Second linker 183 provides attachment between the insulating layer 182
and matrix 184 and consists of the same or similar molecules as first linkers 181. Matrix
layer 184 may consist of a polymer layer, but is also optionally a carbohydrate, protein,

30 poly-amino acid layer or the like. Third linker 185 consists of molecules suitable for
attaching the matrix layer to the antiligand 1 86 and may consist of the same or similar
molecules as either first and/or second linkers 181 and 183.

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Antiligand 1 86 is used to specifically or non-specifically bind the ligand
1 87 within solution and/or to measure physical properties of the solution, some examples
of which are temperature, pH, ionic strength, and the like. Antiligand consists of a
molecule or molecular structure which specifically or nonspecifically binds to ligand 187.
5 For instance, in the case in which the ligand consists of an antigen, antiligand 186 will
consist of an antibody. Ligand 187 consists of a molecule or structure which specifically
or nonspecifically binds to the antiligand 1 86.

Generally, the MBR will be sufficient to interact measurably as described
herein with an electromagnetic test signal along the associated signal path. Thus,

1 0 essentially any MBR composition that exhibits varying dielectric properties can be

analyzed. In most embodiments, the MBR will range in thickness between about 1-5 A to
1 cm. For simple molecular binding events, the range will be usually between about 10 A
to 10,000 A, typically between 100 A and 5,000 A, or 500 A to 1,000 A. In larger
interactions (e.g. cellular) the MBR will range between 1 |im and 100 jim, preferably 5

15 jxm to 50 [im. With insulators, matrices and the like, the size will range significantly
higher.

The embodiment of Fig. ID is not intended to be exhaustive of all possible
MBR configurations. The present invention is not limited to the detection of a molecule
of an anticipated size or structure attached to the signal path. The MBR may consist of 1,

20 2, 3, 4, 5, 10, 20, 30, 50, 100, 1000, or more molecular lengths attached or separated from
but coupled the signal path. Further, the MBR may consist of a multiple layers of
homogeneous molecules, a single but heterogeneous molecular layer or multiple
heterogeneous molecular layers.

Those of skill in the art will appreciate that a vast multiplicity of

25 combinations making up the MBR can be designed, as dictated by the specific

applications. For instance, first, second and third linkers 181, 183, 185, insulating layer
182, and matrix layer 184 are not implemented and the MBR consists of antiligand 186
and ligand 187. Further alternatively, first linker 181 and insulating layer 182 may be
deleted. Other alternative embodiments in which one or more of the described layers are

30 deleted, or additional layers added, will be apparent to one skilled in the art.

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Further, the MBR may be composed of heterogeneous molecules which
may be spatially grouped or randomly layered or distributed depending upon the
particular array format For example, Fig. IE illustrates a top view of an MBR 180
having four different antiligands 190, 191, 192 and 193, which are spatially separated.
Fig. IF illustrates an MBR 180 in which four different antiligands 190, 191, 192 and 193
are randomly distributed throughout. In another embodiment, Fig. 1G illustrates a cross-
sectional view in which the MBR 180 contains cells 194 in solution 157 coupled to signal
path 153. In another embodiment, a cell membrane 195, with membrane bound structures
(not shown), is in solution 157 coupled to signal path 153. The layers may include for
example, linkers, matrices, antiligands, ligands and one or more insulating layers. In
some embodiments, one or more membranes may be employed, such as those controlling
ion transport, size or charge selection or supporting the attachment of antiligand or other
molecular structures.

Electrically, the MBR exhibits unique dielectric properties which are in
part attributable to the structural and conformational properties, and changes therein, of
bound molecules, both isolated and in the presence of environmental changes such as
binding events, pH changes, temperature, ionic strength and the like. The dielectric
properties of the bound molecular structures, along with the local structures of the
solvating medium (the solution) may also be attributable to changes in the intramolecular
and intermolecular bonds caused by primary or other higher-order binding, and the
displacement of the solvating medium near the conductive layer.

Once a conductive layer is provided, one of skill in the art will be
generally familiar with the biological and chemical literature for purposes of selecting a
system with which to work. For a general introduction to biological systems, see,
Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc, and John Wiley & Sons, Inc.
(through 1997 Supplement) (Ausubel); Watson et al (1987) Molecular Biology of the
Gene. Fourth Edition. The Benjamin/Cummings Publishing CO., Menlo Park, CA;
Alberts et al (1989) Molecular Biology of the Cell, Second Edition Garland Publishing,
NY; The Merck Manual of Diagnosis and Therapy. Merck & Co. T Rathway, NJ. Product
information from manufacturers of biological reagents and experimental equipment also
provide information useful in assaying biological systems. Such manufacturers include,
e.g., the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis,

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MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc.
(Palo Alto, CA), Aldrich Chemical Company (Milwaukee, WI), GIBCO BRL Life
Technologies, Inc, (Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka
Chemie AG, Buchs, Switzerland, Applied Biosytems (Foster City, CA), as well as many
5 other commercial sources known to one skilled in the art.

Biological samples can be derived from patients using well known
techniques such as venipuncture, lumbar puncture, fluid sample such as saliva or urine, or
tissue biopsy and the like. When the biological material is derived from non-humans,
such as commercially relevant livestock, blood and tissue samples are conveniently

10 obtained from livestock processing plants. Similarly, plant material used in the invention
may be conveniently derived from agriculture or horticultural sources, and other sources
of natural products. Alternatively a biological sample may be obtained from a cell or
blood bank where tissue and/or blood are stored, or from an in vitro source, such as a
culture of cells. Techniques for establishing a culture of cells for use as a source for

15 biological materials are well known to those of skill in the art. Freshney, Culture of
Animal Cell s, a Manual of Basic Technique. Third Edition . Wiley-Liss, NY (1994)
provides a general introduction to cell culture.

The present invention can be practiced in a number of embodiments.
Some are detailed below, additional embodiments and applications are detailed in the

20 applications section.

In one embodiment, the invention is used to detect binding of a molecular
structure to the signal path. In this embodiment, a signal is propagated along the signal
path. As it propagates, it couples to the bound structure and is modulated. Analysis of
the modulated response indicates binding.

25 In another embodiment, the invention may be used to identify secondary

binding. For example, primary binding may be the attachment of an antibody to the
conductive surface. Secondary binding might involve the measurement of binding
between the immobilized antibody and its antigen in solution. After primary binding has
been detected as described in the previous paragraph, the solution containing the antibody

30 is added to the bio-assay device and the response measured again. The response is

compared to the primary binding response. A change would indicate that a binding event
has occurred.

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In one aspect, the present invention may be used to identify ligands, for
example proteins, in the primary binding stage. In the calibration phase the responses of
a large number of known proteins can be determined and stored. After attaching an
unknown protein to the assay surface, the dielectric properties of the system could be
5 measured and the dielectric properties of the signal used to identify the protein on the
surface. Because each protein's fingerprint response is stored, the unknown response can
be compared with the stored responses and pattern recognition may be used to identify the
unknown protein.

In another embodiment, the invention may be used in an array format. The

10 device will have multiple addressable sites, each of which has bound to it a specific

antiligand. After delivering solution to the device, binding responses at each site will be
measured and characterized. A device of this type may be used to measure and/or
identify the presence of specific nucleic acid sequences in a sample. At each of the
addressable sites a unique nucleic sequence is attached as the antiligand. Upon exposure

15 to the sample, complementary sequences will bind to appropriate sites. The response at
each site will indicate whether a sequence has bound. Such measurement will also
indicate whether the bound sequence is a perfect match with the antiligand sequence or if
there are one or multiple mismatches. This embodiment may also be used to identify
proteins and classes of proteins.

20 In another embodiment, this invention may be used to generate a standard

curve or titration curve that would be used subsequently to determine the unknown
concentration of a particular analyte or ligand. For example, an antibody could be
attached to the device. The device could be exposed to several different concentrations of
the ligand and the response for each concentration measured. Such a curve is also known

25 to those skilled in the art as a dose-response curve. An unknown sample can be exposed
to the device and the response measured. Its response can be compared with the standard
curve to determine the concentration of the ligand in the unknown sample.

In another embodiment, this invention may be used to internally self-
calibrate for losses due to aging and other stability issues. For example with antibody-

30 antigen systems, this invention allows one to measure the amount of active antibody on
the surface by measuring a primary response before exposure the unknown. The value of
the primary response is used to adjust the secondary response, antigen binding, by a
constant that reflects the amount of active antibody that remains on the device.

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IE. The Bio- Assay Device
A. Device Structure

Structurally, the bio-assay device includes a signal path and a bio-
5 electrical interface. The signal path may consist of a single input/output signal port; one
input signal port path and one output port path, or multiple input and/or output signal port
paths. The signal path(s) may be realized in a number of different architectures, such as a
conductive wire, a transmission line, a waveguide structure, resonant cavity, or any other
transmission medium that will support the propagation of the test signal over the desired

10 frequency range. For possible embodiments, see R. E. Collins Foundations for

Microwave Engineering. McGraw-Hill Publishing Co., 1966; and S. March, Microwave
Transmission Lines and Their Physical Realizations. Les Besser and Associates, Inc.,
1986. Further, the bio-assay device may also be realized in a variety of different
configurations. Non-exhaustive configurations include large to miniaturized structures

15 using conventional manufacturing techniques, conventional etching and
photolithography, or semiconductor processing techniques.

Fig. 2A illustrates one embodiment of the bio-assay device as shown in
cross-sectional view. The bio-assay device 230 consists of a top plate 231, contact
terminals 237, and a bottom plate 239. Top plate 231 includes a bottom surface having an

20 signal path 233 disposed thereon. The dielectric substrate 240 and the ground plane 250
are located external to the bio-assay device. Top plate 231 and/or dielectric substrate 240
are formed from an insulating material, such as glass, which are preferably compatible
with conventional photolithography or gold sputtering, etching or chemical vapor
deposition (CVD) processing. Other materials such as alumina, silicon, gallium arsenide

25 or other insulating materials, may alternatively be used.

As illustrated in Fig. 2A, the bottom surface of the signal path 233 is in
contact with the molecular binding region (MBR) 234. As illustrated, the MBR may
consist of bound molecular structures of different layers or types as well as molecular
structures occurring within the solution. In alternative embodiments, the MBR 234 may

30 extend over small or large portions of the signal path 233 and may consist of different
bound molecular structures as shown. The MBR may consist solely of antiligand/ligand
structures, or a variety intermediate of linker, matrix, and insulating layers, as shown in
Fig. ID. When implemented, the insulating layer 182 (Fig. ID) may consist of air,

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polyimide, alumina, diamond, sapphire, or semiconductor insulating material such as
silicon dioxide or gallium arsenide or a non-conductive material in addition to other
conventional insulating materials. The thickness and dielectric constant of the insulating
layer are such that the MBR 234 and the signal path 233 are tightly coupled together
5 during signal transmission. The thickness of the insulating layer 182 may be lO^m, 10" 2
m, 10" 3 m, 10" 4 , 10~ 5 m, KT 6 m, 10' 7 m, 10" 8 m, 10" 9 m, 10* 10 m or less in thickness, or values
ranging therebetween, depending the amount of coupling required, the dielectric constant
of the insulating layer, and the total coupling area. Coupling may be accomplished
through a number of different configurations, including broadside and offset coupled

1 0 configurations in multi-layer, coplanar, or waveguide circuit topologies. Implementing an
insulating layer may be advantageous for hermetically sealing the signal path from the
solution medium and/or for preventing DC or low frequency current from flowing into
the solution which could possibly disrupt molecular binding events occurring therein.

The signal path 233 consists of a material which is capable of supporting

1 5 signal propagation and which is capable of binding the MBR 234. The material will vary
depending upon the makeup of the MBR, but some will include gold, indium tin oxide
(ITO), copper, silver, zinc, tin, antimony, gallium, cadmium, chromium, manganese,
cobalt, iridium, platinum, mercury, titanium, aluminum, lead, iron, tungsten, nickel,
tantalum, rhenium, osmium, thallium or alloys thereof. Alternatively, the signal path 233

20 may include one or more molecular structures (antiligands) (which forms a part of the

MBR 234) for forming bonds with one or more targeted molecules (ligands). The material
comprising the signal path may also be chosen to promote the attachment of linkers as
well as to support signal propagation. Other materials that can be used to form the signal
path 233 will be readily apparent to those of skill in the art.

25 The ligands may be transported to the MBR 234 using a solution 260, such

as Dulbecco's phosphate-buffered saline (d-PBS) for example. The protein, nucleic acid,
or other ligand of interest can be applied to the binding surface using a variety of
techniques such as wicking, pipeting, dipping, dropping, direct contact, or through
capillary action.

30 In a specific embodiment, the signal path 233 is designed to provide low

signal loss and close impedance matching to the external transmission lines 270. Low
signal loss is achieved by fabricating the signal path 233 from a conductive material,
some examples being gold, copper, aluminum, indium tin oxide (ITO) or other

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conductive materials described above. Close impedance matching is achieved by
defining the width of the signal path 233 at approximately the width of external
transmission lines 270, depending on the relative dielectric properties of the substrate, the
solution, and the MBR. Signal continuity between the signal path 232 and the external
5 transmission lines 270 is provided via contact terminals 237. As explained above, the
MBR 234 and solution medium 260 may be located proximate to the ground plane 250
alternatively, or in addition to its location proximate to the signal path 232.

Additional analog and/or digital circuitry in lumped element form,
distributed form, or a combination of both may be included at the input and/or output

10 ports of the bio-assay device. For instance, impedance matching circuits and/or buffer
amplifier circuits may be employed at the input port. Alternatively, or in addition,
impedance matching circuitry and one or more output amplifiers may be implemented to
further enhance the output signal. Those of skill in the art of electronics will appreciate
that other types of conditioning circuitry may be used in alternative embodiments as well.

15 Fig. 2B illustrates a second embodiment of the bio-assay device. In this

embodiment, the solution occupies a space above the signal path 233 which is formed on
the top surface of bottom plate 239. The top side of the signal path 233 forms the binding
surface to which the MBR 234 adheres. Dielectric layer 240 is positioned between signal
path 233 and the ground plane 250. Contact terminals 237 provide a signal path to the

20 external transmission lines 270. The signal path, top plate, bottom plate, contact

terminals, and dielectric layer may be formed from the materials and the processes as
described above. The MBR may also be configured as described above in Fig. ID, or
variations thereof. Further, the MBR 234 and solution medium 260 may be located
proximate to the ground plane 250 alternatively, or in addition to its location proximate to

25 the signal path 233.

Additional structural embodiments include bio-assay devices having multi-
element transmission lines, waveguides, and resonant cavities, in which the MBR may be
attached to one or more of the line or cavity elements in such a way as to enhance
detection specificity and sensitivity. Examples of such structures include parallel

30 arranged signal combiners, resonant cavities, or waveguides along which the bound MBR
on one element alters the signal propagation properties as compared to another parallel
element without the bound structure, and thus serve to change the mode properties of the
combined signal, resulting in readily detectable output signal properties. These latter

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effects make use of well-known techniques to measure frequency, frequency stability, and
very small changes in the frequency with ultra-high precision.

B. Binding Surface Chemistry
5 Fig. 3 illustrates one embodiment of the binding surface chemistry which

occurs along the conductive layer of the bio-electrical interface. The bio-electrical
interface includes a substrate 320, a conductive layer 330, a MBR 340, and solution 350.
The substrate 320 may be any of the dielectric layer or substrate materials described
herein including alumina, diamond, sapphire, plastic, glass and the like and may provide

10 structural support to the conductive layer 320. In an alternative embodiment, substrate
320 is removed and structural support is provided via insulating layer 342.

The conductive layer 330 consists of a material and morphology which
will promote signal propagation over the desired frequencies and which will promote
binding of the MBR 340, as described above. In a two-conductor circuit topology,

1 5 conductive layer 330 may comprise the signal plane or the ground plane. In either case
however, a second conductive layer (either the signal plane or the ground plane, not
shown) is located either below the substrate 320 (the arrangement of Fig. 2B) or at least
one substrate layer removed from the solution 350 (an inverted arrangement of Fig. 2 A).
Alternatively, conductive layers may be positioned at both of these levels.

20 Solution 350 is coupled to the MBR 340 for permitting the flow of ligands

to the MBR 340. Ligand flow from solution 350 to MBR 340 may directionally or non-
directional. Solution consists of any transporting medium such as gases, ligius, or solid
phase materials, some examples being aqueous d-PBS, Tris buffer, phosphate buffers, and
the like.

25 Along the bio-electrical interface, the MBR is positioned between at least a

portion of the solution and the signal path, such that the MBR is more proximate to the
signal path than the solution along that portion. In the embodiment of Fig. 3, the MBR
340 is positioned between the solution 350 and the conductive layer 330, closer in
proximity to the latter. In one embodiment (shown in Fig. 2A), the solution is positioned

30 between the signal and ground planes. In a second embodiment (shown in Fig. 2B), the
solution is positioned outside of the signal-ground plane region.

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The typical chemistry involved in binding the MBR to the conductive
surface will in general depend on the nature and content of the MBR its function in the
assay. The MBR may consist of a ligand, ligand/antiligand complex, or other molecular
structures as described herein. Typically, the ligand will be functionally intact, as close to
5 the surface as possible, and the surface density of the antiligand will be high enough to
provide the greatest dielectric effect, but not so high as to impair the function of binding,
such as by steric hindrance or physically blocking the active binding site of the
immobilized antiligand by neighboring molecules.

Ligands may bind specifically or non-specifically either directly to the

10 . conductive layer 320 or intermediate structures as shown in Fig. 3. If specifically bound
ligands are desired, a linker is optionally used to facilitate the binding, for example to
bind all proteins such that conductive layer 320 is exposed to solution. To ensure a
densely pack binding layer, thiol groups, Fab, or proteins such as protein A may be used
to facilitate the binding of antibodies or other antiligands along the conductive layer 320.

1 5 These and similar substances may be applied to the conductive layer 320 in a number of
ways, including photolithography, semiconductor processing, or any other conventional
application techniques.

In addition, some ligands and antiligands may be able to bind in multiple
ways. These ligands typically have a statistically predominant mode of binding or may

20 be engineered to bind in a site-specific way. Some antiligands optionally bind the surface
in a site-specific manner. For example, an oligonucleotide might be bound at one
terminus. Genrally, the antiligand will be attached in a manner which will not impair the
function of the antiligand, e.g., preferably at concentrations that minimize surface
denaturation.

25 The concentration of the antiligand on the binding surface will vary,

depending upon the specific analyte. For example, typical concentrations for proteins are
10 7 /cm 2 , 10W, 10W, 10 10 /cm 2 , 10 n /cm 2 , 10 12 /cm 2 , 10 13 /cm 2 , 10 14 /cm 2 , 10 l5 /cm 2 , or
concentrations ranging therebetween. Typical concentrations for nucleic acids are
10 7 /cm 2 , 10W, 10W, 10 ,0 /cm 2 , 10 u /cm 2 , 10 12 /cm 2 , 10 I3 /cm 2 , 10 14 /cm 2 , 10 15 /cm 2 ,

30 10 l6 /cm 2 , 10 ,7 /cm\ 10 18 /cm 2 , 10 19 /cm 2 , 10 20 /cm 2 , or concentrations ranging therebetween.
Typical concentrations for analytes in whole blood range from 55M, 25M, 10M, 1M,
.5M, 10" l M, 10" 2 M, 10" 3 M, 10^M, 10" 5 M, 10" 6 M, 10" 7 M, 10* 8 M, 10* 9 M, 10" 10 M, 10" U M,

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l(T l2 M, 10" l3 M, 10~ 14 M, 10" l5 M, 10~ 16 M, 10" l7 M, 10" l8 M, or concentrations ranging
therebetween.

Enough ligand should adhere within the MBR to alter the transmission of a
signal through the bio-electrical interface. The quantity of ligands adhering to the binding
5 surface may consist of 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 n , 10 12 , 10 13 or
more ligands, as well as any number therebetween depending upon the surface area of the
conductive layer. The ligands need not be applied in predefined regions along the
conductive layer since the signal responses are determined by inherent dielectric
properties of the MBR as opposed to placement on the bio-assay device or chip. The

10 MBR will generally have a surface density for smaller molecules ranging from 10 10 cm 2
to 10 24 cm 2 , typically 10 15 cm 2 to 10 20 cm 2 . The ligand layer may be as thin as 1 layer,
but 2, 3, 4, 5 or 10 or more layers are optionally used.

Once a ligand is bound to the conductive layer, the chemistry and/or
structural biology of the system comes into play. The ligand' s dielectric properties yield

1 5 a signal response which is characteristic of the bound structure(s), thereby permitting

binding event detection, as well as detection of other properties of interest in the structure.
The unique response provided by the binding event will depend on the immobilized
antiligand, its target ligand, and the rearrangement of the nearby solution molecules (such
as water and free ions). The range of molecules that can bind to the surface include but

20 are not limited to proteins, nucleic acids, small molecules, saccharides, lipids, and any
other molecule of interest.

Typically, the molecules of the MBR are disposed within a solution which
may consist of an aqueous solution of water, d-PBS, Tris, blood, physiological buffer,
cerebrospinal fluid, urine, sweat, saliva, other bodily secretions, organic solvents, and he

25 like. Other solutions may include gases, emulsions, gels, and organic and inorganic
compounds

The secondary binding reaction occurs at the MBR of the bio-assay device.
A ligand in a solution is transported across the bio-assay device such that it contacts the
antiligand of the binding layer. The concentration of the ligand in the solution varies and
30 may consist of 10' 1 M, 10" 2 M, 10' 3 M, 10" 4 M, 10" 5 M, 10" 6 M, 10" 7 M, 10' 8 M, 10" 9 M, 10"
10 M, 10" 11 M, 10" 12 M, 10- 13 M, 10" 14 M, 10" 15 M, 10' 16 M, 10* 17 M, 10' 18 M, 10" 19 M, 10' 20
M. When an interaction, such as binding, occurs between the ligand and the antiligand,

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the ligand, then optionally becomes part of the binding layer, as dictated by the chemical
equilibrium characteristics of the binding event.

The MBR includes the bound ligands and may also include solution
molecules. The bound ligands can be any molecule, including proteins, carbohydrates,
lipids, nucleic acids, and all other molecules discussed herein. The MBR may further
include a linker to aid in the binding of the antiligand to the binding surface layer.

Additionally, the interaction of the antiligand with the ligand changes the
characteristic dielectric response of the binding layer with only the antiligand attached.
For example, if antiligand A is the antiligand that forms the binding layer, the dielectric
response of a test signal propagating along the transmission line will reflect the
characteristic properties of the structure of antiligand A. When ligand B binds to
antiligand A, the structure and/or dielectric properties of the binding layer will change
due to the binding of A to B. The structure of A may change as B binds to it, thus
providing a different signal response. The change in signal due to the binding interaction
will be characteristic of the binding of A to B. Therefore, the presence of a binding
interaction can be determined from the change in the signal.

Moreover, information about the type of bond or the structural and/or
conformational changes upon binding is obtained by noting which parts of the signal
response have changed due to the interaction. Ligand B is optionally detected and
identified by the signal change upon its binding to antiligand A. The binding of ligand B
to antiligand A induces a conformational change, or other change in the molecular
structure or surrounding solution, in antiligand A and its environs. These changes alter
the dielectric properties of the MBR, thereby altering the signal response of the test signal
propagating along the signal path. The change in the test signal can be used to detect the
ligand B binding event and the particulars of the change can be used to identify the ligand
B. In as much as the relationship between structure and function of the molecule is
known, for example in the case of enzymes, antibodies, receptors and the like, the
function of the bound ligand can be deduced from its spectral identification.

In one embodiment, one type of antiligand is applied to the binding surface
to form a MBR, and a ligand is applied across the MBR to detect a binding event between
the two molecules. In another embodiment, the antiligand may be a mixture and the
ligand that is applied across the binding layer is a known analyte or antibody. By
detecting specific changes in the signal response, the particular ligand with which the

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antiligand interacted can be determined due to conformational and other changes induced
in the ligand or antiligand, and the spectral response resulting therefrom. Such an
embodiment does not require the spatial isolation of each of the specific antiligands, but
rather derives the desired level of specificity from the spectral response, so that a given
5 binding interaction is determined by looking at the electromagnetic response rather that
noting on which part of the assay the binding event took place.

In another embodiment, the antiligand may be a known molecule on the
binding layer and the ligand applied across the bio-assay device as a mixture of
unknowns, such as a whole blood sample. In this case, the presence of a particular ligand

10 such as an antibody in the blood is detected by the presence or absence of a particular
peak or signal in the spectrum that results from passing a signal through the bio-assay
device. Alternatively it can be detected due to the changes in the spectrum of the
antiligand or ligand upon binding of the ligand. Such an embodiment increases the
specificity of the detection over that of the binding chemistry alone, since the signal

1 5 contains information about the nature of the binding event. Thus, specific binding may be
distinguished over non-specific binding, and the overall specificity of detection may be
greatly improved over the specificity of the chemistry alone.

The system of detection formed through use of the bioassay device
provides a high throughput detection system because detection optionally occurs in real

20 time and many samples can be rapidly analyzed. The response period is optionally

monitored on a nanosecond time scale. As soon as the molecules are bound to each other,
detection occurs. More time is optionally required to measure low concentrations or
binding events between molecules with a low binding affinity. The actual time is
optionally limited by diffusion rates. Other than these potential limitations, thousands of

25 compounds are optionally run through the system very quickly, for example, in an hour.
For example, using chip fab technologies, a 10,000 channel device (using some of the
emerging microfluidics technologies) is possible, and with small volumes and thus short
diffusion times, and kinetic measurements measuring only the beginning of the reaction,
10 million samples per hour are optionally measured. With known concentrations, the

30 binding affinity is optionally calculated from the kinetics alone and thus the device can be
probed at a very fast time scale and the affinity calculated and/or estimated from the slope
of the kinetic curve. References for kinetics and affinities can be found in any standard

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biochemistry or chemistry text such as Mathews and van Holde, Biochemistry. Benjamin
Cummings, New York, 1990.

C. Bio-Electrical Interface
5 The bio-electrical interface is the structure along which the MBR and the

signal path are formed. As described above, the signal path may consist of a conductive
or dielectric waveguide structure, a two conductor structure such as a conventional
signal/ground plane structure, or three or more conductor structures known in the art.
Generally, the thickness of the conductive region of the signal path is designed to provide

1 0 minimal signal loss. For example, a typical thickness of gold transmission line is in the
order of 0.1 to lOOOjim, preferably about 1-10 ^m.

The signal path is formed along a direction which is non-orthogonal to the
MBR. In one embodiment, the test signal propagates in parallel to a tangent on the
surface on which the MBR is formed. In other embodiments, the test signal may

15 propagate at an angle of ± 1°, ± 2°, ± 3°, ± 4°, ± 5°, ± 10°, ± 15°, ± 20°, ± 30°, ± 40°, ±
45°, ± 50°, ± 60°, ± 70°, ± 80°, or ± 85° relative to the MBR binding surface, or any
ranges therebetween. In a first embodiment, the signal path consists of a transmission
line in a two conductor structure and the direction of the signal path is defined by the
Poynting vector as known in the art of electromagnetics. In a second embodiment, the

20 transmission line may consist of a conductive region or layer which extends continuously
along the bio-electrical interface region. In a third embodiment, the signal path maybe
defined as the path having the least amount of signal loss along the bio-electrical interface
over the desired frequency range of operation. In a fourth embodiment, the signal path
maybe defined as having an a.c. conductivity of greater than 3 mhos/m, i.e., having a

25 conductivity greater than that a saline solution, typically greater than 5 mhos/m, but
ideally in the range of 100 to 1000 mhos/m and greater..

The operation of the bio-electrical interface will be better understood by
developing an equivalent circuit model for the interface. The equivalent circuit models
presented are shown in a two-conductor circuit topology, although those of skill in the art

30 of circuit design will readily appreciate that each may be implemented in single conductor
waveguide topologies, resonant circuit topologies, as well as circuit topologies with three
or more conductors.

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Fig. 4A illustrates one embodiment of an equivalent circuit model 420 for
the bio-electrical interface structure shown in Fig. 2A. Those of skill in the art of circuit
design will appreciate that the illustrated circuit model is not exhaustive and that other
equivalent circuit models may be derived from the bio-electrical interface of Fig. 2 A.
5 The illustrated equivalent circuit model includes series blocks 422a, 424a,

and 426a which models the series electrical effects of the signal path 232, the MBR 234,
and the solution 260, respectively, all as shown in Fig. 2A. The signal path, MBR, and
solution circuit blocks 422a, 424a, and 426a are coupled in parallel since the signal path,
the MBR, and the solution, each provides a possible longitudinal signal path along the

1 0 interface. In an alternative embodiment where the MBR and solution are located

proximate to the ground plane, the signal path and the ground planes of the equivalent
circuit model 420 are switched. In the embodiments where the MBR and solution are
located proximate to both the signal path and the ground plane, Fig 2A represents the top
half of the equivalent circuit, the bottom half (ground plane) of which is identical if the

1 5 same solution and MBR is used.

The equivalent circuit model 420 further includes shunt circuit blocks
422b, 424b, and 426b which models, respectively, the shunt electrical effects of the
dielectric layer 240, the MBR 234, and the solution 260, shown in Fig. 2A. The series
orientation of shunt blocks 422b, 424b, and 426b results from the physical arrangement of

20 each of these elements, occurring serially from signal path through the MBR, solution,
and the dielectric layer, to the ground plane, the arrangement of which is shown in Fig.
2A.

Fig. 4B illustrates one embodiment of a circuit 430 corresponding to the
equivalent circuit model shown in Fig. 4A. Those of skill in the art of circuit design will

25 readily appreciate that other circuits configurations are possible. The series circuit blocks
422a, 424a, and 426a each consists of a series-coupled resistor and inductor. The shunt
circuit blocks 422b, 424b, and 426b each consists of a parallel-coupled resistor and
capacitor. Series resistors R t , R m , R s model respectively the resistivity of the signal path,
the MBR, and the solution. Shunt resistors Rm', IV, R<j model respectively the resistivity

30 of the MBR, the solution, and the dielectric layer. Series inductors Lt, Lm, Ls model
respectively the inductance of the signal path, the MBR, and the solution. Shunt
capacitors C m , C s , C d model respectively the capacitances of the MBR 234, the solution
260, and the dielectric layer 240. Collectively, the aforementioned resistors, inductors,

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and capacitors define the circuit 430 which transforms the input signal Vj into the output
signal V 0 .

The dielectric properties of the MBR largely determine the values of the
circuit elements corresponding to each of those layers. For instance, in the illustrated
5 embodiment of Fig. 4B, the susceptibility of the MBR largely defines the value of the
shunt capacitance C m . Further, the dispersive properties of the MBR largely determine
the value of the shunt resistance R m \ The values of C m and R^' define to a significant
degree the signal response of the bio-electrical interface. Thus, the signal response of the
bio-electrical interface is strongly characteristic of the dielectric properties of the MBR

1 0 and can be used to detect and identify molecular binding events, as will be further
described below.

In embodiments where the solution 260 is an aqueous solution, the
dielectric properties associated therewith are disadvantageous to signal propagation along
the signal path. Specifically, water and other highly aqueous solutions such as whole

15 blood, exhibit a relatively high resistance Rs and a relatively low resistance R/, as well as
absorptive properties with respect to electromagnetic radiation in certain areas of the
spectrum. The magnitude of these parameters results in very high signal loss along the
signal path. The location of the MBR between the signal path and the solution in the
present invention serves to insulate from, or otherwise modulate the coupling with, the

20 signal and the solution, thereby modulating the signal loss and changing other parameters
of signal propagation.

Fig. 4C illustrates one embodiment of an equivalent circuit model 450 for
the bio-electrical interface structure shown in Fig. 2B. Those of skill in the art of circuit
design will appreciate that the illustrated circuit model is not exhaustive and that other

25 equivalent circuit models may be derived from the bio-electrical interface of Fig. 2B.

The illustrated equivalent circuit model 450 includes series and shunt
circuit blocks 452a and 452b which electrically model the signal path. The equivalent
circuit model 450 also includes a MBR circuit block 454 coupled in series with a solution
circuit block 456 which electrically models the MBR and solution. As explained above,

30 the orientation of the series and shunt blocks 452a and 452b define a conventional

transmission line structure. Additionally, the series orientation of the MBR and solution
circuit blocks 454 and 456 results from signal field lines extending from signal path,
through the MBR, and into the solution, the arrangement of which is shown in Fig. 2B.

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Alternative circuit models may be derived as above for bio-assay devices implementing a
MBR and solution proximate to the ground plane alternatively, or in addition to their
location near the signal path.

Fig. 4D illustrates one embodiment of a circuit 470 corresponding to the
5 equivalent circuit model shown in Fig. 4C. Those of skill in the art of circuit design will
readily appreciate that other circuits configurations are possible. The series and shunt
circuit block 452a and 452b collectively represent the conventional model for the signal
path. The MBR circuit block 454 is coupled between the signal path and the solution
circuit block and in one embodiment, consists of a parallel coupled capacitor C m , resistor

10 R m and inductor Lm. Collectively, the aforementioned resistors, inductors, and capacitors
define the circuit 470 which transforms the input signal Vj into the output signal V 0 .

As explained above, the dielectric properties of the MBR and solution will
affect the values of each of the electrical elements. In particular, the susceptibility and .
other dielectric properties of the MBR will largely determine the value of Cm ; the

1 5 permittivity, other dielectric properties, and surface morphology of the MBR will strongly
define the value of Lm; and the dispersive properties as well as conductive and other
dielectric properties of the MBR will significantly determine the value of R m . Thus, the
signal response of the bio-electrical interface is strongly characteristic of the dielectric
properties of the MBR and can be used to detect and identify molecular binding events, as

20 will be further described below.

D. Specific Embodiments

Figs. 5A-5G illustrate specific embodiments of the bio-electrical interface
implemented in a two conductor circuit topology. Those of skill in the art of circuit
25 design will readily appreciate that each may be implemented in a single conductor
waveguide topology, as well as three or more conductor circuit topologies.

Each of the embodiments consists of a signal plane 520, dielectric layer
530, and a ground plane 550. Coupled to signal plane 520, ground plane 550 or both are
a MBR 5 1 5 and a solution 5 1 0. In each of the embodiments, the MBR 520 may either be
30 in direct contact with the signal path 530, or coupled thereto. When the signal plane 520
contacts the MBR 515 directly, it is formed from a material which is capable of both
supporting signal propagation and adhering ligands, such as proteins, nucleic acids,
carbohydrates, enzymes and the like. Such materials include, but are not limited to gold,

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ITO, copper, silver, zinc, tin, antimony, gallium, cadmium, chromium, manganese,
cobalt, iridium, platinum, mercury, titanium, aluminum, lead, iron, tungsten, nickel,
tantalum, rhenium, osmium, thallium or alloys thereof. Other materials which can be
used will be readily apparent to those of skill in the art.

The dielectric layer 530 may consist of air, polyimide, teflon, woven
insulating materials such as Duriod™, alumina, diamond, sapphire, or semiconductor
insulating material such as silicon dioxide or gallium arsenide, or other insulating
materials. The thickness and dielectric constant of the dielectric layer 530 are selected to
provide the desired transmission line impedance as known in the art. The solution 510
may consist of any transporting medium, such as Dulbecco's phosphate-buffered saline
(d-PBS), which provides the subject molecular structure. The protein, nucleic acid, or
other ligand of interest can be added to the bio-electrical interface using a variety of
techniques such as wicking, pipeting or through capillary action.

Figs. 5A and 5B illustrate cross-sectional views of the interface realized in
a microstrip circuit topology and in which the solution 510, and MBR 515 are positioned
above and below the signal path 530, respectively. Figs. 5C and 5D illustrate cross-
sectional views of the interface in which the solution 510 and MBR 51 5 are positioned
above and below the ground plane 550, respectively.

Fig. 5E illustrates a cross-sectional view of the interface realized in
coplanar waveguide topology. In this embodiment, the solution 510 and MBR 515 are
positioned above the signal path 530. Alternatively, the solution 510 and MBR 515 may
be positioned below the signal path 530, or above or below one or both of the coplanar
ground planes 550. Fig. 5F illustrates a cross-sectional view of the interface realized in a
stripline circuit topology. In this configuration, the solution 5 10 and MBR 5 15 are
positioned above the signal path 530. In other embodiments, these layers may
alternatively or in addition be place below the signal path 530, or above or below one or
both ground planes 550.

Fig. 5G illustrates one embodiment of the bio-electrical interface
implemented in a coaxial circuit topology, A first insulator 530a having a cavity 570
partially circumscribes an interface center conductor 530. The MBR 515 is positioned in
proximity to the uncovered portion of the interface center conductor 530. A second
insulator 540b is provided between the outer conductor 550 and the first insulator 540a

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and circumscribes the outer conductor 550, forming the cavity 570 in which the solution
510 resides. The radii and dielectric constants of the first and second insulators 530a and
530b may be of the same or differing values and each is selected to provide the desired
line impedance and the requisite measurement sensitivity over the test signal frequency
5 range. In an alternative embodiment, the MBR 5 15 is located proximate to the outer
conductor 550. In this embodiment, the second insulator 530b includes a cavity for
allowing the MBR to form proximate to the outer conductor and the first insulator
completely circumscribes the center conductor 320. Further alternatively, the MBR 5 1 5
and solution may be located outside of the outer conductor 550.

1 0 The bio-electrical interface may be fabricated in a variety of shapes

depending upon the application, for example, squares, ellipsoids, rectangles, triangles,
circles or portions thereof, or irregular geometric shapes, such as one that would fit into
the bore of a hypodermic needle. The size of the bio-electrical interface will vary
depending upon the application and have sizes on the order of 10m 2 , lm 2 , 10' V, lO'V,

15 10- 3 m 2 ,10-W, 10" 5 m 2 , lO^m 2 , 10- 7 m 2 ,10- 8 m 2 , 10"V, 10- 10 m 2 , 10- n m 2 ,10" 12 m 2 , or range
anywhere therebetween, The bio-electrical interface may be fabricated to fit into
something as small as a needle bore. The interface may alternatively be modified to
accommodate other diagnostic applications, such as proteomics chips. The size or shape
of the bio-electrical interface need only be such that signal propagation and molecular

20 binding therealong is possible.

The signal path included within the bio-electrical interface region supports
the propagation of an electromagnetic signal at the desired test frequency. Many signal
path configurations are possible, one example being a sputtered gold transmission line
operable between D.C. and 110 GHz. In another embodiment, the signal path consists of

25 a dielectric medium, such as the MBR itself. In this embodiment, the signal path blocks
DC voltages and currents but otherwise supports the propagation of the desired test
signal, occurring at frequencies, for instance 1 MHz, 5 MHz 10 MHz, 20 MHz, 45 MHz,
80 MHz, 100 MHz, 250 MHz, 500 MHz, 750 MHz, 1 GHz, 2.5 GHz, 5 GHz, 7.5 GHz, 10
GHz, 12 GHz, 18 GHz, 20 GHz, 22 GHz, 24 GHz, 26 GHz, 30 GHz, 33 GHz, 40 GHz,

30 44 GHz, 50 GHz, 80 GHz, 96 GHz, 100 GHz, 500 GHz, 1000 GHz, or frequencies
ranging therebetween. Accordingly, the signal path is designed using high frequency
circuit design techniques, known in the art. Such design techniques include impedance
matching the signal path to the interconnecting structures, minimizing the insertion loss

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of the signal path, and minimizing the Voltage Standing Wave Ratio (VSWR) of the
signal path. In the preferred embodiment of the present invention, the signal path and
MBR are oriented in a non-orthogonal orientation.

The present invention is not limited to the detection of a molecule of an
5 anticipated size or structure attached to the signal path. The MBR may consist of 1,2, 3,
4, 5, 10, 20, 30, 50, 100, 1000, or more molecular lengths attached or separated from but
coupled the signal path. Further, the MBR may consist of a multiple layers of
homogeneous molecules, a single but heterogeneous molecular layer or multiple
heterogeneous molecular layers.

V. Measurement Methodology
A. General Overview

The measurement methodology of the present invention makes use of the
observation that a vast number of molecules are distinguishable from one another based

1 5 upon their unique dielectric properties which include dispersion effects, resonance effects,
and effects on the solution surrounding said molecules. In the present invention, when a
test signal couples to the MBR, the MBR interacts with the energy of the test signal,
resulting in a unique signal response. The unique signal response can then be used to
detect and identify the molecules which make up the MBR.

20 Those of skill in the art will appreciate that most molecules exhibit

variation in dielectric properties over different frequencies. For instance, a molecule may
exhibit a dramatic change in its dielectric properties as a function of frequency in one or
more regions of the electromagnetic spectrum. The frequency band over which the
molecule exhibits a dramatic dielectric change is often referred to as the molecule's

25 dispersion regime. Over these regimes, the molecule's dielectric constant, permittivity,
dipole and/or multipole moments, and susceptibility will change dramatically as a
function of frequency. These quantities are often complex, having both real and
imaginary parts to account for both the magnitude and phase changes that occur in the
signal response. The dispersion regimes range over various frequencies, including the RF,

30 microwave, millimeter wave, far-infrared, and infrared frequencies.

The molecule's dielectric properties can be observed by coupling a test
signal to the molecule and observing the resulting signal. When the test signal excites the
molecule at a frequency within the molecule's dispersion regime, especially at a resonant

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frequency, the molecule will interact strongly with the signal, and the resulting signal will
exhibit dramatic variations in its measured amplitude and phase, thereby generating a
unique signal response. This response can be used to detect and identify the bound
molecular structure. In addition, because most molecules will exhibit different dispersion
5 properties over the same or different frequency bands, each generates a unique signal
response which can be used to identify the molecular structure.

Detection and identification of molecular binding events can be
accomplished by detecting and measuring the dielectric properties at the molecular level.
The dielectric properties at the molecular level can be defined by the molecule's
10 multipole moments, the potential energy of which can be represented as an infinite series
as is known in the art:

- , x q p-x 1 ^ x t Xj

V r L U r

The infinite series consists of multiple terms, each of which describes in varying degrees
15 the molecule's dielectric properties in the presence of an electric, magnetic or an electro-,
magnetic field. The first term is referred to as the monopole moment and represents the
scalar quantity of the electrostatic potential energy arising from the total charge on the
molecule. The second term or "dipole moment" is a vector quantity and consists of three
degrees of freedom. The third term or "quadrapole moment" is a rank-2 tensor and
20 describes the molecule's response over 9 degrees of freedom. In general, the N th term is
a tensor of rank N-l, with 3 N_1 degrees of freedom, though symmetries may reduce the
total number of degrees of freedom. As one can appreciate, the higher-order moments
provide greater detail about the molecule's dielectric properties and thus reveals more of
the molecule's unique dielectric signature. Since the gradient of the potential results in
25 the electric field:

£ = -VG>(x),

The field strength of the higher-order moments falls off rapidly as a
30 function of distance and thus their contribution is difficult to measure. For instance, the
field due to dipole moment falls off as r" 3 and the field due to the quadrupole moment

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falls off as r" 4 . Thus, this approach requires close proximity between the binding
molecules and test signal path and low signal loss therebetween. Since it is often the case
that molecular binding event detection occurs in strongly signal-absorbing solutions, such
as whole blood samples or ionic solutions, signal loss between the binding events and
5 signal path becomes quite high and detection of the higher order moments is very
difficult.

In addition, each multipole term couples to the electric field in a different
way. This is demonstrated by first looking at the energy of a given electrostatic system:

10 W= J/7(x)<D(x)d 3 jc

Expanding the electrostatic potential in a Taylor Series gives

<D(x) = 0(0) + x • vo(0) + \YL x > x j irrr

2t / j uX i OXj

Since E = -V0> (x),

d E,

a>(x) = O>(0)-x-E(0)-ISIx i ^-i

Further, for the external field, V • E = 0 , so that we get

O(x) = O(0)-x.E(0)4EZ(3¥y^ 2 ^)^
0 / j ox i

Inserting this back into the equation for the energy given above yields

20 W = ? a>(0)-p.E(0)4ZZ^^

6 i j ax t

This shows the manner in which each multipole term interacts with the
interrogating field: The total charge q with the potential, the dipole p with the electric
field, the quadrupole Qy with the gradient of the electric field, etc. This illustrates the
25 second difficulty with the detection of the higher order multipole moments: It is difficult

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in a bulk sample to achieve sufficient field gradients to couple to the higher order
moments.

The present invention overcomes the aforementioned obstacles by
implementing the described bio-electrical interface. The interface includes a MBR which
5 is coupled along the signal path. The MBR consists of a very thin and highly
inhomogeneous region (from a dielectric standpoint), thus providing the required
proximity to the electromagnetically probing structure as well as the sufficient field
gradients to couple to the higher order multipole moments. These qualities enable
detection of higher order moments which provide a greatly enhanced view of the

10 molecule's dielectric properties. The positioning of the MBR proximate to the signal
and/or ground planes serves to isolate the signal propagating thereon from becoming
absorbed into solution, thereby reducing the signal loss and enabling the usage of higher
test frequencies to more accurately detect and identify the binding events. In this manner,
the present invention enables to a greater degree the recovery or the signal response

1 5 including the contributions from the molecule's dipole and other higher-order multipole
moments.

Using the described bio-assay device of the present invention, numerous
properties associated with the MBR may be detected. Fig. 6A illustrates one embodiment
of this method. Initially at step 602 a MBR is formed and coupled along a portion of a

20 signal path. As described, the MBR may consist of a ligand, antiligand/ligand complex,
etc and be in direct or indirect physical contact with, or electromangentically coupled to
the signal path. The signal path may consist of the signal plane or ground plane in a two-
conductor transmission topology.

Next at step 604, a test signal is propagated along the signal path. The test

25 signal may be any time-varying signal of any frequency, for instance, a signal frequency
of 10 MHz, or a frequency range from 45 MHz to 20 GHz. Next at step 606, the test
signal couples to the MBR and in response develops a signal response to the coupling.
The signal response is then recovered and provides information as to one or more
properties of the molecular binding region.

30 The bio-assay device may used to provide information about numerous

properties of the MBR, such as the detection and identification of molecular binding
events, ligand concentrations, changes in dielectric properties of the MBR, classification
of detected binding events, and the like. In addition, the bio-assay device includes a self-

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calibration capability which is useful in point-of-use quality control and assurance. Each of
these methods and capabilities are further described below. Based upon the described
methods and structures, modifications and additional uses will be apparent to those skilled in
the art.

5 The ability to detect and measure molecular dipole, quadrupole, and higher order

multipole moments in solution represents a significant advance in the art for a number of
reasons. First, many molecules of biomedical interest such as proteins have very distinct
structures, and therefore distinct multipole moments. Thus identifying the multipole
moments for a given molecule reveals properties of said molecule which are unique, and

10 thus allows identification of said molecule. Second, structure and function are intimately
related in many molecules of biomedical relevance, such as proteins. Thus, the ability to
detect properties of a given molecule which relate directly to the function of said
molecule means that functionality may be monitored for whole ranges of activities. Third,
the local physiologic environment often plays an important role in the structure and

15 function of a given molecule, so that an ability to detect the physical properties described
above means that molecules may be used a monitors and probes for the purpose of
measuring changes in a given system. Thus, with the ability to translate complex and
informative properties about molecular and cellular systems into a detectable electronic
data format, whole new possibilities emerge in the areas discussed herein.

B. Detecting Bound Molecular Structures

The bio-assay device described herein enables the detection of molecular
binding events occurring along the signal path. Detectable binding events include
primary, secondary, and higher-order binding events. For instance, in a two-conductor

25 bio-electrical interface having no pre-existing MBR, the molecules of the conductive
layer will form the antiligands for binding to the ligands, the ligands forming the MBR.
In another embodiment, the antiligand and ligand are both included in the MBR. In this
embodiment, the MBR is attached to the signal path surface via linkers, matrix molecules,
insulating layers or a combination of each as show in Fig. ID.

30 Fig. 6 A illustrates one embodiment of this process. Initially at step 602, a

signal path is formed from a material which can support the propagation of a signal over
the desired frequency of operation. The signal path may consist of a single port path, a
two port path, or a multiple port path within one of the bio-assay devices described

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herein. In addition, the signal path may be realized as a transmission line, resonant
cavity, or as a waveguide structure.

Next at step 604, a solution is provided which contains the subject
molecule or molecular structure. At step 606, a MBR consisting of the ligand is formed
5 from the solution and is coupled between at least a portion of the signal path and the

solution. Next at step 608, a test signal is propagated along the signal path. Alternatively,
the test signal may be launched during the application of the solution in order to observed
in real time the signal response occurring as a result of the binding events. At step 610,
the test signal propagates over, couples to the MBR and develops a signal response which
10 indicates the presence of the ligand. Next at steps 612 and 614, the test signal is
recovered, the response of which indicates detection of the ligand.

The dielectric properties of the MBR may contribute to induce any number
of signal responses, each of which may be indicative of molecular binding. For instance,
the dispersive properties of the MBR may vary dramatically over frequency. In this
15 instance, the test signal response will exhibit large changes in the amplitude and/or phase
response over frequency when molecular binding events occur along the binding surface,
thereby providing a means for detecting molecular binding events along the binding
surface.

In another embodiment, the dielectric relaxation properties of the MBR
20 will vary as a function of pulse period of the input signal. In this instance, the test signal
response will indicate a change in the amount of power absorbed, or change in some other
parameter of the test signal like phase or amplitude, at or near a particular pulse period.
By observing a change in the absorbed power or other parameters, binding events along
the binding surface may be detected. Other quantities such characteristic impedances,
25 propagation speed, amplitude, phase, dispersion, loss, permittivity, susceptibility,
frequency, and dielectric constant are also possible indicators of molecular binding
events.

The above-described method may be used to detect the primary binding of
an antiligand or ligand directly or indirectly along the signal path. Similarly, the process
30 of Fig, 6A may also be used to detect secondary binding of a ligand to an antiligand. The
method of Fig. 6 A is not limited to detection of primary or secondary binding events
occurring along the signal path. Indeed, tertiary, and higher-order binding events

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occurring either along the signal path or suspended in solution can also be detected using
this method.

Fig. 6B illustrates a second process for detecting secondary and higher-
order binding events occurring either along the signal path. Initially at step 620, the
5 primary binding event is detected and the signal response measured, one embodiment of
which is shown in steps 602-612. Subsequently at step 622, the primary binding event
signal response is stored and used as a baseline response. Next at step 624, a second
molecular solution is added to the bio-assay device and allowed to circulate over the
binding surface. Next at step 626, steps 608 through 612 of Fig. 6A are repeated to obtain

10 a second signal response. Next at step 628, the second signal response and the baseline
response are compared. Little or no change indicates that the two signal responses are
very close, indicating that the structural and dielectric properties of the MBR have not
been altered by the addition of the molecules within the new solution. In this case,
secondary binding has not occurred to a significant degree (step 630). If the comparison

15 results in a change outside of a predetermined range, the structure and/or dielectric
properties of the MBR have been altered, thereby indicating secondary binding events
(step 632). Quantities which can be used to indicate secondary binding events will
parallel the aforementioned quantities, e.g., amplitude, phase, frequency, dispersion, loss,
permittivity, susceptibility, impedance, propagation speed, dielectric constant as well as

20 other factors. Tertiary or high-order binding events may be detected using this approach.

An alternative method of detecting secondary or higher order binding
events does not required prior knowledge of the specific primary binding event. In this
embodiment, the bio-assay device is designed in the assay development stage to operate
with known parameters, so that whenever a pre-defined change in one of these parameters

25 , is detected, for example at the point-of-use, the binding event or events are then known to
have occurred. In this embodiment, the pre-measurement of a primary binding event is
not necessary, as the initial characterization has already been done either at the time of
fabrication or at the time of design.

Secondary binding events can also be achieved by detecting changes in the

30 structure of the primary bound molecule. When a molecule becomes bound, it undergoes
conformational and other changes in its molecular structure relative to its unbound state.
These changes affect the primary binding molecule's dielectric properties as well as
inducing changes in the surrounding solution, the variation of which can be detected

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using steps 620-628 of Fig. 6B, described above. Quantities which can be monitored to
indicate a change in the dielectric properties of the primary bound molecule include the
aforementioned quantities, e.g., amplitude, phase, frequency, dispersion, loss,
permittivity, susceptibility, impedance, propagation speed, dielectric constant as well as
5 other factors.

C. Detecting Changes in the Dielectric Properties of the Molecular binding region
The bio-assay device described herein may also be used to measure the
dielectric changes of the MBR as a result changes in temperature, pH, ionic strength and
10 the like.

Fig. 6C illustrates an exemplary embodiment of the process. The process
closely parallels the disclosed method for identifying binding events, the exception being
that the method allows for the detection and quantitation of changes in dielectric
properties of the MBR.

1 5 The process begins at step 64 1 , when a solution having an initial dielectric

property is added to the bio-assay device, the signal response is measured and recorded.
In one embodiment, this step is performed according to steps 602-612. After a
predetermined time or operation, a second measurement is made and a second signal
response is recorded (step 642), again in one embodiment according to steps 602-612. At

20 step 643, a comparison is then made between the first and second signals to determine
whether the two signals correlate within a predefined range. If so, the properties of the
solution are deemed to not have undergone any dielectric changes (step 644).

If the signal responses do not correlate within a predefined range, one or
more dielectric properties of the solution is deemed as having undergone (step 645).

25 Optionally the change in dielectric properties may be quantitated in the following manner.
At step 646, the second signal is stored and correlated to a known signal response. The
closest correlated response will identify the dielectric property of the solution and the first
signal response can be correlated to the initial value of the dielectric property, the
difference of which can be used to determine the amount by which the identified

30 dielectric property has been altered (step 647).

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D. Identifying Bound Molecular Structures

Using the described bio-assay devices, it is possible to characterize a
known ligand and subsequently identify it in a solution having an unknown ligand make-
5 up. Fig. 6D illustrates one embodiment of this process. Initially at step 652, a large
number of molecular structures are measured and their responses stored using one or
more of the measurement systems, described below. In one embodiment, this step is
performed according to steps 602-612. Each stored response may correspond to a single
ligand occurring within the solution or multiple ligands occurring within the same

10 solution. Subsequently at step 654, a measurement is made of an unknown solution. In
one embodiment, this step is performed according to steps 602-612. Next at step 656, the
signal response of the solution is compared to the stored signal responses to determine the
degree of correlation therewith. At step 658, the unknown molecular structure is
identified by selecting the stored response which exhibits the closest correlation to the

15 unknown response. The comparison may be performed using one or more data points to
determine the correlation between one or more stored responses, and may involve the use
of pattern recognition software or similar means to determine the correlation. The process
may be used to identify primary, secondary or higher-order bound molecular structures.

20 E. Identifying Classes of Bound Molecular Structures

It is also possible to characterize known molecular sub-structures such as
domains or other structural homologies that are common to similar classes of proteins or
sequence homologies in nucleic acids. In one embodiment, the process proceeds as
shown in Fig. 6D, except that in step 652, N number of molecular sub-structures are

25 measured and their responses stored. Each stored signal response may correspond to one
or more sub-structures. The process continues as described in steps 654, 656 and 658
until a sufficient number or structures have been detected and characterized to identify the
unknown compound. Once a sufficient number of correlations occur, it is then possible to
classify the unknown molecular structure.

30 Fig. 6E illustrates another process by which unknown ligands may be

classified. The process identifies the unknown ligand by detecting binding to structural
motifs on the unknown compound. Initially, at step 660 a bio-assay device is provided
which has multiple addressable arrays, each of which has a antiligand for a specific ligand

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sub-structure. Next at step 662, the presence of particular sub-structures is detected by
the binding of each to its respective antiligand, and subsequent characterization. In one
embodiment, this step is performed according to steps 602-612. Subsequently at step
664, each of the binding events is then characterized by identification of qualities such as
5 affinity, kinetics, and spectral response. At step 666, a correlation is then made between
the known and unknown responses. If each of the unknown responses correlates to
known responses, the ligand is identified as the ligand corresponding to the known
response. If the sub-structures exhibit both correlated and uncorrected responses, the
correlated responses may be used to construct a more general classification of the
10 unknown ligand. This process may be used to identify any molecular structure, for
example proteins, which occur within the same class or have re-occurring structural
homologies.

It is also possible that an intensive spectral analysis of a given unknown
compound could lead to insights on structure and function, as comparisons can be made
15 to known structures, and extrapolation will lead to some level of classification.

A. Specific v.s. Non-Specific Binding:

Specific ligand binding is distinguished form non-specific binding by the
spectral fingerprint of the binding event. A given binding event of interest, for example

20 antibody binding to antigen, may be first characterized in a purified solution containing
just the ligand of interest and the antiligand specific to said ligand on the MBR. A broad
spectral study is then carried out to see when in the spectrum the strongest responses are
found. The assay is then repeated in the solutions typically found in the dedicated
applications, for example whole blood, to determine what effects non-specific binding has

25 on the response. Then various points are found which are determinate of specific binding,
and a separate set of points are found which are determinate of non-specific binding, an a
subset of these frequency points are chosen for the actual assay application. By
comparing the response due to specific binding with those due to the non-specific
binding, the extent of specific binding can be determined.

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B. Characterization of a Given Ligand:

Often it is desirable to determine certain qualities of a given molecule.
Examples in include determining the class to which a protein belongs, or which type of
5 polymorphism a given gene or other nucleic acid sequence is. This may be done in a
number of ways. Proteins are often classified by number and types of structural
homologies, or particular substructures which are found in the same or similar classes of
proteins. For example, G-Proteins commonly found in cell membranes and which mediate
signal transduction pathways between the extra-cellular environment and the intra-

1 0 cellular environment, always have a structure which traverses the cell membrane seven
times. Such a structure is virtually definitive of a G-Protein. Other classes of proteins
have similar structural homologies, and as such, any method which can distinguish one
class of proteins from another on the bases of these homologies is of enormous use in
many of the biomedical research fields. Given that the dielectric properties of a given .

15 molecule is determined entirely by the geometry of the charge distribution of said

molecule, and further given that most proteins have a unique structure or geometry, then
each protein may be uniquely determined by measuring the dielectric properties of the
protein. Thus a simple dielectric signature, such as the ones generated by the present
invention, may serve to uniquely identify a given protein, and further, may allow

20 classification of the protein into some previously known class of proteins. A further
refinement may be added to the classification methodology by using a group of
antiligands on the bio-assay device which are specific for particular sub-structures of a
given protein. For example, a group of antibodies which are specific for particular sub-
structures such as domains may be utilized for the determination of the existence or

25 absence of said sub-structures. Thus, any given protein may be characterized by
determining both the presence and absence of certain sub-structures as well as the
dielectric properties of the protein itself. Further refinements to this classification
strategy may include looking at temperature, pH, ionic strength, as well as other
environmental effects on the above-mentioned properties.

30 Nucleic acids may also be characterized by following a similar paradigm.

For example, a given gene may be known to have a certain base pair sequence. Often
times in nature there will be small variations in this sequence. For example, in the gene
which codes for a chloride ion transport channel in many cell membranes there are

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common single base-pair mutations, or changes. Such changes lead to a disease called
cystic fibrosis in humans. Thus characterizing a given nucleic acid sequence with respect
to small variations is of enormous importance. Such variations are often called
polymorphism's, and such polymorphism's are currently detected by forming
5 complementary strands for each of the known polymorphism's. Since any given gene
may take the form of any one of hundreds or even thousands of polymorphism's, it is
often an arduous task to generate complementary strands for each polymorphism. Using
the invention described herein, non-complementary binding or hybridization may be
detected and distinguished by measuring many of the same physical properties as were

10 described in the previous paragraph: The dielectric properties of the hybridization event
can be characterized and correlated to known data, thereby determining the type of
hybridization which has occurred — either complete or incomplete. Thus with an
antiligand comprised of a given nucleic acid sequence, hundreds of different
polymorphisms (as ligands) may be detected by the characterization of the binding event.

1 5 One of skill in the art will appreciate that further refinements are possible, such as
modifying the stringency conditions to alter the hybridization process, or varying the
temperature and determining the melting point, which serves as another indicator of the
nature of the hybridization process.

In a similar manner, drug-receptor interactions may be characterized to

20 determine is a given binding event results in the receptor being turned on or off, or some
other form of allosteric effect. For example, a given receptor may be used as an
antiligand, and a known agonist may be used as the first ligand. The interaction is then
characterized according to the dielectric response, and this response is saved.
Subsequently, compounds which are being screened for drug candidates are then

25 observed with respect to their binding properties with said receptor. A molecule which
binds and yields a similar dielectric response is then known to have a similar effect on the
receptor as the known agonist, and therefore will have a much higher probability of being
an agonist. This paradigm may be used to characterize virtually any type of target-
receptor binding event of interest, and represents a significant improvement over current

30 detection strategies which determine only if a binding event has occurred or not. Those of
skill in the art will readily appreciate that there are many other classes of binding events
in which the present invention can be applied.

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Examples of sub-structures which may be used in the above method
include: Protein secondary and tertiary structures, such as alpha-helices, beta-sheets,
triple helices, domains, barrel structures, beta-turns, and various symmetry groups found
in quaternary structures such as C 2 symmetry, C 3 symmetry, C 4 symmetry, D 2 symmetry,
5 cubic symmetry, and icosahedral symmetry. [ G. Rose (1979), Heirarchic Organization of
Domains in Globular Proteins, J. Mol BioLl34: 447-470] Sub-structures of nucleic acids
which may be analyzed include: sequence homologies and sequence polymorphisms, A,
B and Z forms of DNA, single and double strand forms, supercoiling foirns, anticodon
loops, D loops, and Tvj/C loops in tRNA, as well as different classes of tRNA molecules. [
1 0 W. Saenger (1 984) Principles of Nucleic Acid Structure. Springer- Verlag, New York; and
P. Schimmel, D. Soil, and J. Abelson (eds.) (1979) Transfer RNA. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N. Y.]

F. Quantitating Concentrations

15 The bio-assay devices described herein may also be used to quantitate the

concentrations of ligands. Fig. 6F illustrates one embodiment of this process. In the
event the device is not precailbrated (step 679), initially at step 670, antiligands are
chosen having the appropriate binding properties, such as binding affinity or kinetics, for
the measured analyte. These properties are selected such that the antiligand's equilibrium

20 constant is near the center of its linear operating region. For applications where the range
of concentration is too wide for the use of a single antiligand, several antiligands may be
used with differing affinities and/or linear operating ranges, thereby yielding a value for
the concentration over a much wider range.

Next at steps 672 and 674, the antiligands are attached to the bio-assay

25 device or chip and the device is connected to the measurement system. At step 674, a
decision is made as to whether the response requires characterization for maximum
specificity. If so, a spectral analysis is performed in which the frequencies where analyte
binding has maximal binding is determined (step 675a), the regions where the non-
specific binding has maximal effect is determined (step 675b), and the unique response

30 due to analyte binding is determined (step 675c). If characterization is not required, or if
so, after its completion, the device is calibrated. This step is performed in one
embodiment by supplying a known concentration of ligands to the bio-assay device and

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measuring the resulting response (step 676a). Alternatively, if more data points are
needed for the calibration (step 676b), then a sample may be chosen with a different
concentration (step 676c), and the response against this concentration may be measured
(step 676a). In one embodiment, the measurement is made in accordance with steps 602-
5 612. Subsequently at step 677, an extrapolation algorithm is generated by recording the
calibration points from the foregoing response. Next at step 678, a sample of unknown
ligand concentration is measured. This step is accomplished in one embodiment by
supplying the unknown sample to the bio-assay device, correlating the response to the
titration algorithm, and determining therefrom the ligand concentration.

10 In the event that a given bio-assay device is either pre-calibrated, or

calibrated by design, the only step required is to apply the ligand or analyte to the surface,
and measure the response. Such a bio-assay device may be realized in many different
ways. For example, some circuit parameter like impedance or characteristic frequency of
a resonant circuit may be designed to change in a pre-determined way when the binding

15 event occurs, and the amount by which the parameter changes may further be designed to
have a dose-response. Thus, a measurement of said circuit parameter will, when analyzed
via a suitable algorithm, immediately yield a quantitative value for the concentration of a
given analyte or ligand.

20 G. Bio-assay Device Self-Calibration

The described bio-assay devices possess a self-diagnostic capability and thus
a point-of-use quality control and assurance. For a given dedication application, a particular
antiligand (primary binding species) will act as an antiligand for some ligand (the
secondarily binding species) of interest in the solution. The primary binding species may be

25 attached at the point of fabrication, and the secondary binding species may be attached at the
point-of-use. Thus, variations in fabrication — especially the attachment of the primary
species — will cause variations in the ability of the device to bind its specific ligand.
However, the amount of ligand bound will be in direct proportion to the amount of
antiligand bound, thus a ratiometic measurement of the two is possible.

30 Fig. 6G illustrates one embodiment of the process. Initially at step 680, a

molecular binding surface is formed along the signal path by binding the appropriate
antibody at various concentrations and characterising the resulting response for each of
these concentration, yielding some value "x" for each concentration. Next, at step 682, a

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similar titration curve is generated for the ligand by measuring the antibody/ligand
binding response for several different concentrations of ligand, and a ligand titration
curve is pre-determined. Next, at step 684 a scale factor A is generated by taking the ratio
of responses of antibody binding to ligand binding. At the point-of-use, the uncalibrated
assay is then first probed (step 686) to determine the amount of bound antibody "x" and
the scale factor "y" resulting therefrom. The ligand is then applied to the assay and the
response is measured (step 689), and the response and predetermined titration curve are
scaled by the scale factor "y" (step 690) to determine unknown concentration.

The process of Fig. 6F may also be modified to allow quantitating the
amount of ligand in the solution. In the modification, the binding surface of the bio-assay
device includes antiligands having a predefined affinity and ligand specificity. The solution
is subsequently applied to the device, and a response is measured. The signal response will
be proportional to the amount of the ligand that has bound. Thus, a titration of any given
ligand may be carried out by choosing an antiligand with an appropriate linear operating
range — the range in which the equilibrium constant is within a couple of log units of the
desired range of concentrations to be detected. The same ratiometic analysis as described
above can be applied to yield a robust and precise quantitative assay with internal controls
and calibration necessary to insure reliability.

Each of the described methods may be practiced in a multitude of different
ways (i.e., software, hardware, or a combination of both) and in a variety of systems. In
one embodiment, the described method can be implemented as a software program.

Fig. 7 A illustrates an example of a computer system 710 for executing a
software program designed to perform each of the described methods. Computer system
710 includes a monitor 714, screen 712, cabinet 718, and keyboard 734. A mouse (not
shown), light pen, or other I/O interfaces, such as virtual reality interfaces may also be
included for providing I/O commands. Cabinet 718 houses a CD-ROM drive 716, a hard
drive (not shown) or other storage data mediums which may be utilized to store and
retrieve digital data and software programs incorporating the present method, and the like.
Although CD-ROM 716 is shown as the removable media, other removable tangible
media including floppy disks, tape, and flash memory may be utilized. Cabinet 71 8 also
houses familiar computer components (not shown) such as a processor, memory,, and the
like.

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Fig. 7B illustrates a simplified system block diagram of a typical computer
system 710 used to execute a software program incorporating the described method. As
shown in Fig. 7 A, computer system 710 includes monitor 714 which optionally is
interactive with the I/O controller 724. Computer system 710 further includes subsystems
5 such as system memory 726, central processor 728, speaker 730, removable disk 732,
keyboard 734, fixed disk 736, and network interface 738. Other computer systems
suitable for use with the described method may include additional or fewer subsystems.
For example, another computer system could include more than one processor 728 (i.e., a
multi-processor system) for processing the digital data. Arrows such as 740 represent the

10 system bus architecture of computer system 710. However, these arrows 740 are

illustrative of any interconnection scheme serving to link the subsystems. For example, a
local bus could be utilized to connect the central processor 728 to the system memory
726. Computer system 710 shown in Fig. 7B is but an example of a computer system
suitable for use with the present invention. Other configurations of subsystems suitable

1 5 for use with the present invention will be readily apparent to one of ordinary skill in the
art

V. Measurement Systems

Various measurement systems may be used to perform the above-
20 described methods. Figs. 8-10 illustrate three examples of possible measurement
systems: a frequency domain test system, a time domain test system and a dielectric
relaxation measurement system.

A. Frequency Measurement System

25 Fig. 8A illustrates one embodiment of a frequency measurement system in

accordance with the present invention. The system 800 includes a signal source 810
coupled to the bio-assay device input 852 and a signal detector 890 coupled to the bio-
assay device output 858. Optionally, an additional signal source may be coupled to the
bio-assay device output 858 and an additional signal detector coupled to the test circuit

30 input 852 for providing complete two-port measurement capability. The system may be
modified to a one-port test system in which a signal detector is coupled to the signal path
for receiving a reflected signal. In a specific embodiment, the aforementioned frequency
measurement system consists of a network analyzer such as model number 85 10C from

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the Hewlett-Packard Company. Other high frequency measurement systems, such as
scalar network analyzers, which provide signal information based upon transmitted and
reflected signals may alternatively be used.

Measurements are made according to the aforementioned methodologies.
5 Initially, an incident signal 860 is launched toward the test circuit and the transmitted
and/or reflected signals 870 and 890, respectively, are subsequently recovered. The
resulting signal responses will take the form of unique frequency responses or "spectral
fingerprints," two examples of which are shown in Figs. 8B and 8C. Fig. 8B illustrates one
type of frequency response in which a resonance occurs at frequency f res . Here, response

10 870 undergoes a steep fall and rise, indicating little or no signal energy reaches the output
port at this frequency. The resonance is caused by the dielectric property and impedance
of the MBR changing over frequency f start to f stop . Different ligands will resonate at
different frequency points. In addition, some ligands may exhibit multiple resonant
frequency points over the measured band f sta rt to f s t 0 p. Once a ligand has been

15 characterized as having one or more uniquely occurring resonance points, this data can be
used to identify the presence of the ligand in an unknown solution. This characterization
can be ascertained from empirical data or from theoretical calculations of multipole
moments and resonant frequencies. Furthermore, when detecting the presence of
secondary binding events, this data can indicate when an analyte is bound to a ligand by a

20 change in the one or more unique resonance points.

Fig. 8C illustrates another type of frequency response which can be used to
detect or identify a molecular structure. In this case, the frequency response exhibits a
generally monotonically increasing or decreasing trend with some degree of amplitude
variation. The response's slope and/or the amplitude variation may be used to detect

25 and/or uniquely characterize the bound molecule. Thus in the described manner, the
resonant frequency points, slope, trend, and variation of the test signal's phase may be
used to uniquely identify the molecular binding event. The frequency response may be
measured at the input port 852, at the output port 858 or at both ports to uniquely identify
the bound molecular structure.

30 Fig. 9 illustrates a second exemplary embodiment of a frequency

measurement system in accordance with the present invention. The bio-assay device
under test 920 consists of coaxial topology (shown in Fig. 5G) having a center conductor
921, a first insulator 922 having a cavity 922a, a second insulator 923, and an outer

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conductor 924. Solution 926 occupies cavity 922a. Of course, devices of other circuit
topologies may be tested as well.

Once the solution 926 is added to the cavity 922a, the molecules within the
solution 926 form a MBR 92 1 a proximate to the center conductor 92 1 . During the
5 measurement, a signal source 910 launches an incident test signal 912 to center conductor
921. The MBR 922a modulates the incident test signal 912, and the reflected test signal
932 provides a unique signal response which can be used to identify the ligand. The one-
port coaxial configuration may be realized, for instance, as a sub-cutaneous needle
structure.

B. Time Domain Measurement System

Fig. 10 illustrates one embodiment of a time domain measurement system
1000 in accordance with the present invention. The system includes a pulse source 1002
and a detector 1004 coupled to the test circuit input 1022. In an alternative embodiment,

15 an additional pulse source and detector may be coupled to the output port 1028 to

provide complete two-port measurement capability. Further alternatively, the system may
comprise a one-port test system in which a signal detector is coupled to the signal path for
receiving a reflected signal. In a specific embodiment, the time domain measurement
system consists of a time domain reflectometer such as model number 1 1 801

20 manufactured by the Tektronix Corporation. Other high frequency measurement systems,
such as network analyzers having a time domain measurement mode which provide signal
information based upon transmitted and reflected signal pulses may alternatively be used.

In the time domain measurement system, the input test signal 1060
consists of a time domain pulse, the reflected portions of which can be displayed over

25 time. In the present embodiment, an incident pulse 1060 is launched toward the portion
of the transmission line which is tightly coupled to the assay surface. Due to the
dielectric property of the MBR, a portion of the incident pulse 1060 is reflected toward
the detector 1004. The reflected pulse 1070 will exhibit a unique shape and/or time delay
which is characteristic of the MBR's dielectric properties, which are in turn largely

30 defined by the dielectric properties of the ligand, antiligand, and the surrounding solution.
Thus, the pulse shape and delay of the reflected pulse 1070 can be used to characterize
and identify the ligand. The time domain test system may be used separately or in
conjunction with the high frequency test system to identify one or more unknown ligands.

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C. Dielectric Relaxation Measurement System

As known in the art, the dielectric relaxation frequency of a ligand is the rate
at which the dielectric properties of the molecular level changes when an electric field is
5 applied to the molecule. As with the dielectric properties of the ligand, the dielectric

relaxation frequency is primarily defined by the structure and binding geometries unique to
each molecule. Thus once measured, the dielectric relaxation frequency of a ligand can be
used to identify it.

The dielectric relaxation frequency can be quantified by measuring the rate at

1 0 which the ligand absorbs power over frequency. Fig. 1 1 illustrates one embodiment of a
system 1 100 for making this measurement. The measurement system 1 100 is similar to the
time domain measurement system 1000 illustrated in Fig. 10 and includes a pulse source
1 102 and a detector 1 104 coupled to the test fixture input 1 122. An additional pulse source
and detector may be coupled to the output port 1 128 to provide complete two-port

15 measurement capability. In a specific embodiment, the time domain measurement system
consists of a time domain reflectometer such model number 1 1801 manufactured by the
Tektronix Corporation. Other high frequency measurement systems, such as network
analyzers having a time domain measurement mode which provide signal information
based upon transmitted and reflected signal pulses may alternatively be used.

20 The input test signal 1 160 consists of separate pulse groups, each group

having two or more incident pulses and a different pulse interval. The pulse groups 1 162
and 1 1 64 are launched toward the portion of the transmission line which is tightly
coupled to the binding surface. If a pulse group 1 162 has an interval substantially
equivalent to the dielectric relaxation period (the reciprocal of the relaxation frequency),

25 the MBR will absorb successively less energy in succeeding pulses. The decrease in

signal absorption can be measured in the reflected response 1 170 at the input port 1 122 or
at the output port 1 128. As an alternative measurement quantity, the remaining signal
power may be measured either at the input port 1 122 or the output port 1 128.

The rate of change of signal absorption and the pulse interval at which the

30 change occurs can then be plotted and used to characterize and identify the unknown
bound molecule(s). This system characterization may be used independently or in
conjunction with the above-described time and/or frequency domain test systems.

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In all of the above systems, one of skill in the art will readily appreciate
that such systems can be scaled down to the chip level using such technologies as
Microwave Monolithic Integrated Circuits (MMIC) and the like. Such miniaturized
systems can be readily extended to highly parallel systems capable of detecting and
5 measuring hundreds, thousands, or tens of thousands of compounds simultaneously.

These systems can be configured to yield "logic gates" which are switched by the binding
event itself, such as by changing a characteristic impedance and thus the transmission
and/or reflection coefficients, or by changing the band pass properties of such a circuit,
and using this as the on/off gate.

VI. Examples

A. Example 1 : Detection of a ligand binding to the surface.

Primary binding of urease to an ITO surface was demonstrated in the bio-
assay device as shown in Fig. 2A. The binding surface of the bio-assay device comprised

15 a cover glass treated with ITO deposited via chemical vapor deposition (CVD). The ITO
transmission line was carefully examined to ensure that it contained no microfractures or
breaks in it. The transmission line was measured with a Tektronix 1 1801 signal analyzer
with a TDR module, and found to have a broadband reference impedance of 32 fi. The
line length was about 2.6 nsec in length, the binding surface was found to have an

20 impedance of 34 Q, and a length of about 200 psec. Separation between the top and
bottom plates were 10 mils, and the chamber was long. No side walls were used;
instead, the capillary action of the top and bottom plates retained the solution in place.

Next, the bio-assay device filed with a solution of d-PBS, With the bio-
assay device filled, baseline transmission loss (S21) and return loss (S\\) S-parameter

25 measurements were made over a test frequency range from 45 MHz to 1 GHz. The

measurements were made and stored using a network analyzer model number HP 851 0B
from the Hewlett-Packard Company. Next, urease was added in a volume excess of 10:1 . ■
Transmission loss and return loss S-parameter measurements was repeated and compared
to the baseline measurement.

30 Table 1 below shows these values for 100 MHz and 1 GHz and the return

loss and transmission loss measurement responses are shown in Figs. 12A and 12B. The
data indicates that the bio-assay test fixture exhibited a return loss (Si 1) change of - .5 dB

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and -0.42 dB, respectively at 100 MHz and 1 GHz between the d-PBS filled chip and the
d-PBS + protein filled device. The fixture exhibited a transmission loss change (S 2 i) of
+.325 dB and +.450 dB at 100 MHz and 1 GHz, respectively.

To determine if the signal responses were due to a bulk effect (proteins in
5 solution), or to proteins binding to the binding surface, each response was recorded and
the protein solution was flushed with d-PBS in a volume excess of 25:1 (2 mL of d-PBS
to .075 mL chamber size). The bio-assay device was then re-measured from 45 MHz to 1
GHz as described above.

As can be seen from comparing the last two columns of Table 1, the effect
10 of flushing the protein from the bio-assay device had minimal effect on the return loss
and transmission loss measurements. This indicates that the measured effect was indeed
due to the urease binding the binding surface within the bio-assay device. In general, it
was noted that the replacement of the solution containing the ligand with an identical
solution without the ligand caused very little or no change in the response.

15 Table 1

The Effect of Primary Binding of Urease

Frequency

Protein in Solution

After d-PBS Flush

100 MHz

-500 milli-dB

-475 milli-dB

1 GHz

-420 milli-dB

-200 milli-dB

S21

100 MHz

+325 milli-dB

+300 milli-dB

1 GHz

+450 milli-dB

+400 milli-dB

B. Example 2: Identification of Collaeenase and Lvsozvme through Primary Binding
Using a bio-assay device similar to the one cited in example 1 above, and

20 prepared and characterized in a similar manner, we carried out a series of experiments to
examine the differing responses of different proteins over the frequency range of 1-
10GHz. The same device was used for each experiment (to eliminate small differences in
fabrication from one device to another), but was thoroughly washed with SDS between
the application of each of the proteins. Figs. 12C and 12D illustrate the transmission loss

25 measurements of the primary binding effects of collagenase and lysozyme samples,

respectively, over the test frequency range from 1 GHz to 10 GHz. In both instances, the
signal response exhibited a pattern of peaks and valleys which can be used to detect and
identify the ligand uniquely. In particular, the frequency response of the collagnase

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sample exhibited a strong positive peak near 5 GHz. The response of the lysozyme
sample indicated a relative flat response near 5 GHz and a strong positive peak near 8
GHz. For each of the other numerous proteins examined, the response was unique to each
protein, and readily allowed identification of an unknown protein within the group. Of
5 course, additional spectral points may also be compared and analyzed to distinguish these
and other molecular substances. The responses may be stored and later recalled to
identify unknown samples. In addition the less-pronounced peaks may be examined
collectively to determine patterns for particular ligands.

10 C. Example 3: Detection of Secondary Binding: Concanavalin A to Dextran

This application provides an example of secondary binding detection,
using a bio-assay device similar to the one cited in example 1 above, and prepared and
characterized in a similar manner. Concanavalin A (con- A) is a glucose binding protein
that can be found in jack beans, and was used as the primary binding antiligand The con-

15 A used here was obtained from Sigma Chemical Company. Dextran, a glucose

polysaccharide, was then used as a ligand to bind con-A, with glucose as a competitive
means of reversing the dextran binding to demonstrate specificity. (Dextran and glucose
were also obtained from Sigma Chemical Company.)

The transmission line was the same as that discussed in Example 1, with a

20 nominal 32 Q reference impedance, and an ITO cover glass with a DC resistance of 80 Q
and a nominal TDR impedance of 34 Q. A concentration of approximately 15 |iM
solution of con-A was placed directly into the bio-assay device, and allowed to reached
equilibrium. Evaporative losses did not dry out the chamber as established by visual
inspection. After the system was flushed and stabilized, dextran was added to bind the

25 con-A. After a change in the signal was detected, the chamber was flushed with 10 mg/ml
d-PBS and the signal response was measured a second time. This effect is shown in
Figure 12E at 1GHz. The unbound response being used as the baseline response. As
shown, the bounded response appears to be .25 dB less lossy than the unbound response.
Binding specificity was confirmed by competing off the bound dextran with glucose,

30 followed by a d-PBS flush to free the glucose. The latter step returned the signal to the
baseline obtained before the dextran had been added to the device, thus demonstrating
specificity of the binding event.

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D. Example 4: Detection of Small Molecule Binding.

Using a bio-assay device similar to the one cited in example 1 above, and
prepared and characterized in a similar manner, the bio-assay test fixture and network
5 analyzer set-up was used to demonstrate that small molecules binding to large molecules
may also be detected with the present invention. In order to probe the bio-assay device at
higher frequencies, the device was reproducibly and carefully placed in a Faraday box to
shield it from external influences. This allowed the device to be probed at frequencies up
to 20GHz. Initially, con-A was added into the bio-assay device and allowed to bind to the

10 bio-electric interface. A transmission loss measurement was made, stored, and used as
the baseline response 1252 as shown in Fig. 12F.

Next, a glucose concentration of 10 mg/ml was added to the bio-assay
device and used to bind the con-A antiligand. A transmission loss measurement was
made and plotted relative to the baseline response 1252 to determine the change in signal

15 response due to small molecule binding.

As can be seen from Fig. 12F, the binding response 1254, which
corresponds to the binding of glucose to con-A, is distinguishable from the baseline
measurement 1252. In particular, the binding response 1254 exhibits 2 large peaks
between 16-20 GHz which is not observed in the baseline response 1252. The difference

20 in the measured signal responses 1252 and 1254 provides the basis for detecting when
glucose has bound to the con-A antiligand. This was followed by a flush with the d-PBS
buffer only, and the response was reversed as the bound glucose dissociated from the con-
A. A separate experiment looking at the effect of glucose on the bare chip (i.e. no con-A
as an antiligand) showed that glucose alone has little if any effect on the response to

25 electromagnetic interrogation in the above mentioned frequency spectrum, thus showing
that the result shown is due entirely to the effect of glucose binding to con-A.

The experiment was repeated for biotin binding to avidin. Avidin was
added to the bio-assay device as the antiligand, and a transmission loss measurement was
made, stored and used as a baseline response 1262. Next, a lpM concentration of biotin

30 was added, and a transmission loss measurement was made relative to the baseline
measurement. The results are shown in Figure 12G.

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The binding response 1264 corresponding to the biotin bound to Avidin
indicates a deep null between 14-16 GHz and a large peak near 20 GHz. The differences
between the baseline response (indicating unbound Avidin) and the binding response
1264 (indicating bound Avidin) is dramatic and can be used to detect the bound Avidin
5 molecule.

E. Example 5: Quantitation titrations

These experiments demonstrate that the magnitude of the signal change
upon a ligand binding to an antiligand is a function of the number of sites that are

10 occupied. The test system using a bio-assay device similar to the one cited in example 1
above, and prepared and characterized in a similar manner, was used with dextran
binding to con- A, with glucose used as a competitive inhibitor. A series of dilutions was
created that centered around the binding constant of con-A. Dextran as an antiligand was
bound to con-A such that 100% binding occurred, A series of competing glucose

15 concentrations was used to compete off the dextran, so that the concentration of dextran
on the molecular binding surface was commensurably decreased.

The standard transmission line configuration as discussed above was used.
Con-A was bound to the molecular binding region and the system was stabilized. The
bio-assay device was then flushed with d-PBS and data obtained at 1GHz. The results of

20 this competition titration are shown in Figure 12H. The results show how the signal

changes as the concentration of glucose is increased from 0 to 15 mg/dl. The signal of the
Con-A changes as the dextran is released and the glucose is bound (which actually
measures the avidity of the dextran). Specificity was also demonstrated by reversal by
glucose of the dextran binding effect.

25 Table 2 shows the magnitude of the change in transmission loss as a

function of the glucose concentration for some selected concentrations.

Table 2

Dextran fully bound

+320 milli-dB

lmg/ml glucose

+280 milli-dB

1.33 mg/ml glucose

+275 milli-dB

2 mg/ml glucose

+240 milli-dB

5 mg/ml glucose

+115 milli-dB

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lOmg/ml glucose

-5 milli-dB

A simple glucose titration was also carried out at a resonant point in the
spectrum of con- A. Figure 121 shows the change in the return loss as a function of
glucose concentration at this resonance point, demonstrating two effects: First, glucose
5 has a dose-response effect as a ligand which is based on the effect it has on the antiligand
(which in this case is con- A). Second, there are regions in the spectra which show a much
more sensitive response to the ligand/antiligand binding event than other regions.

A succession of serial dilutions of the dextran solution which took the
concentration down below one picomolar (10.i 5 Molar) showed that even at these low
10 concentrations, a significant signal response indicating binding occurred. The time

required for the accumulation of the signal ranged from several minutes to ten minutes,
but the response was characteristic of the detection of dextran at higher concentrations.

F. Example 6: Detection of Nucleic Acids

15 In order to demonstrate the ability to detect nucleic acids, a bio-assay

device with polylysine as the antiligand attached to a gold surface was fabricated. Using a
bio-assay device similar to the one cited in example 1 above except for the gold surface,
and prepared and characterized in a similar manner, a high concentration solution (about
20uM) of calf-thymus DNA was prepared in a d-PBS buffer. The polylysine was placed

20 on the bio-assay device, and the transmission loss response was measured. The response
was checked for stability over time and saved. The chamber was then flushed with the
buffer, the response again checked for changes with the flush and stability thereafter, and
the response stored as the baseline response.

A solution containing the DNA was then placed in the bio-assay device,

25 and the change in the response was measured by subtracting the resulting response from
the baseline response, and observed for stability. The bio-assay device was flushed with
buffer to remove the DNA in the bulk, leaving only the DNA/Polylysine complexes on
the bio-assay device surface. The resulting change is shown in Fig. 12J.

G. Example 7: The Effects of pH and Salinity

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The effects of pH and salinity in the signal were measured in two different
experiments. To investigate the effects of the pH, a series of buffers or pH ranging from
3.94 to 9.80 were measured. The 60 Hz conductivity for each buffer was measured to
correct for the change in free ions. Subsequently, transmission loss responses at 100MHz,
1GHz, and 10GHz was measured. The results are shown in Fig 12K.

A similar experiment was carried out to determine the effects of changing
the ionic concentration of a solution. Several solutions were made, starting with a simple
d-PBS, and adding various amounts of sodium chloride. The 60Hz conductivity was then
measured and noted, and the samples were serially placed in the bio-assay device and the
transmission response was measured at 100MHz, 1GHz, and 10GHz. These results are
plotted in Figure 12L.

As both of these plots show, certain environmental changes result in
changes in the measured parameters.

H. Example 8: Detection in Whole Blood:

The detection of troponin-I (TN-I) was made in whole, unprocessed
human blood was made to verify detection capability in messy environments. The
unprocessed human blood was treated with sodium citrate to anticoagulate. An anti-TN-I
antibody corresponding to the epitope of TN-I was used for calibration purposes. The
signal path of the bio-assay device was coated with anti-TN-I Ab (antiligand). A sample
of blood was spiked to a lOng/ml concentration of TN-I and a second identical sample of
blood was left unspiked as a control.

The experiment consisted of attaching the anti-TN-I Ab antiligand to the
device; then first running the unspiked sample across the device; flushing the sample
chamber several times to see what the noise of exchange was; followed by the spiked
sample, which was also replaced several times to establish a noise floor. In each case, the
change in the transmission loss was measured. As a check, the anti-TN-I Ab antiligand
was removed from the device. The experiment was subsequently repeated as a control to
determine if any other properties of the two blood samples (assumed identical except for
the TN-I spike) were responsible for the change. The following table shows the result of
this experiment for a probe signal at 1GHz.

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Control
Anti-TN-I

Unspiked sample

Spiked Sample

<20miIli-dB

<20 milli-dB

+275 milli-dB

In a second series of experiments, ten different samples of blood were
obtained from a clinical laboratory, untreated except for being anticoagulated with
heparin. One of the samples was divided into two parts, and one of the parts was spiked
5 with the TN-I antigen as described in the previous paragraph. The bio-assay device was
then prepared with the anti-TN-I antibody on the surface. Each sample was then serially
passed through the bio-assay device, saving the spiked sample for last. The responses for
each of these samples, probed at 1GHz as in the previous experiment, and shown in Fig.
12M. The spiked sample was clearly distinguishable form the rest of the (unspiked)
10 samples.

I. Example 9: Detection of the Ouadrupole Moment of a Molecule

The effect of binding avidin to a gold surface was investigated to
determine the detectablity of a molecule's quadupole moment. Avidin is a tetramer
15 which has a very small dipole moment in the unbound state owing to the symmetry of the
molecule in the unbound state. Figure N shows the result of avidin binding with
characteristic peaks as shown in the plot. Note that these peaks are markedly smaller than
the peaks which arise due to the binding of biotin, as shown in Fig. 12G.

20 VTI. Applications

The methods and systems of the present invention may be used in a variety
of applications, examples of which are described herein.

The present invention could be used to quantitate the level of binding
between a ligand and an antiligand and thus be used to determine the effect of other
25 molecules on the activity of an enzyme. For instance, other molecules in the solution
could decrease or increase the level of the binding and thus the identity of enzyme
inhibitors or inducers could be determined.

The presence of infectious pathogens (viruses, bacteria, fungi, or the like)
or cancerous tumors can be tested by monitoring binding of an antiligand to the pathogen,

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tumor cell, or a component of the pathogen or tumor, such as a protein, cell membrane,
cell extract, tumor markers like CEA or PSA, other antigenic epitopes or the like. For
example, the invention is capable of detecting the pathogen or tumor by detecting the
binding of pathogenic or tumor markers in the patient's blood with an antibody on the
5 bio-assay device. In addition for example, the binding of an antibody from a patient's
blood to a viral protein, such as an HIV protein is a common test for monitoring patient
exposure to the virus. Another common example is the quantitation of Prostate Specific
Antigen (PSA) in patient blood as a marker for the progression of prostate cancer.

Additionally, drug receptor interactions, including both membrane and

1 0 non-membrane receptors and receptor conformational changes as a result of drug binding
can be determined with the present invention. In another aspect, the invention can be
used to provide information on lipid interactions, such as lipo-proteins binding to lipids,
and liposomal interactions with lipids.

In additional embodiments, the technology of the invention can be used to

1 5 provide gene chips for screening nucleic acid samples and proteomics chips for

cataloging and describing proteins. Such chips can make use of the unique ability of the
invention to measure simultaneously the affinity, kinetics, and unique dielectric
signatures of each binding event; and to make these measurements at a multiplicity of
addressable test sites on the chip. The exact nature of the addressing will depend on the

20 applications, but the general strategy is as follows: Define a vector space by the variables
K^q, k A , and co=(o)l,co2,a)3,...) where these variables represent the equilibrium constant,
the kinetic constant, and a basis set of N frequencies at which the dielectric properties are
probed. An N+2 dimensional space is thus defined into which every binding event can be
mapped. A group of reference molecules is subsequently chosen which represents a

25 spectrum of binding events of interest, such as a group of oligonucleotides with different
nucleic acid sequences or a selection of antibodies which are specific for protein domains
or other sub-structures of proteins, and attach them to addressable points on the chip. A
particular species of molecules or group of species is introduced to the chip, and each
address is then probed for the value of each of the points in the vector space defined

30 above (or a suitable subset thereof). Each species can then be represented by an address
in the vector space. The complexity of the system will depend on the size of the vector
space and the total number of different immobilized ligands on the surface.

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As an example of the above, consider a simple system comprised of two
different nucleic acid probes which are analyzed at four different frequencies; and further,
each of these frequencies can be parsed into ten different amplitudes. Such a system
would have 100 million possible addresses (10 4 for the first polymorphism and 10 4 for the
5 second polymorphism). An unknown placed in the system will be represented by a unique
address of the form [(1,5,3,7)(4,8,6,7)], where the first four numbers represent the
spectral response of the first probe at the four selected frequencies, and the latter four
numbers represent the spectral response of the second probe at the four selected
frequencies. Thus with just two probes and four frequencies, 100 million unique

1 0 addresses can be generated.

"Smart Needle" IV assays, which provide a miniature bioassay device in
the bore of a needle, can also be made to use the technology of the present invention.
This embodiment can be used to provide cost-effective and safe medical diagnostics
devices for use in emergency rooms and other points-of-care settings and the like.

15 Examples of uses include: diagnosing acute conditions such as heart attacks, infectious
diseases like bacterial meningitis or Group B Step infections in the neonatal/perinatal
setting, coagulopathies, fetal and neonatal oxygenation in the intensive care setting;
diagnosing chronic conditions in point-of-care settings such as health care provider
offices and remote locations.

20 A bio-assay device bearing a plurality biological binding partners permits

the simultaneous assay of a multiplicity of analytes in a sample. In addition, the
measurement of binding of a single analyte to a number of different species of biological
binding partners provides a control for non-specific binding. A comparison of the degree
of binding of different analytes in a test sample permits evaluation of the relative increase

25 or decrease of the different analytes.

The bio-assay device of this invention can be used to detect virtually any
analyte in vivo or ex vivo. While in a preferred embodiment the analyte may be a
biological molecule, it need not be so limited so long as a specific binding partner is
available or some other property of the analyte can be measured in some embodiment of

30 the invention described herein. Suitable analytes include virtually any analyte found in

biological materials or in materials processed therefrom. Virtually any analyte that can be
suspended or dissolved preferably in an aqueous solution can be detected using the
methods of this invention. Examples of analytes of interest include 1) antibodies, such as

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antibodies to HIV 2), Helicobacter pylori, hepatitis {e.g., hepatitis A, B and C), measles,
mumps, and rubella; 2) drugs of abuse and their metabolic byproducts such as cotinine,
cocaine, benzoylecgonine, benzodizazpine, tetrahydrocannabinol, nicotine, ethanol; 3)
therapeutic drugs including theophylline, phenytoin, acetaminophen, lithium, diazepam,
5 nortryptyline, secobarbital, phenobarbitol, and the like; 4) hormones and growth factors
such as testosterone, estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid
stimulating hormone, follicle stimulating hormone, luteinizing hormone, transforming
growth factor alpha, epidermal growth factor, insulin-like growth factor I and II, growth
hormone release inhibiting factor, and sex hormone binding globulin; and 5) other

10 analytes including glucose, cholesterol, caffeine, corticosteroid binding globulin, DHEA
binding glycoprotein, and the like.

As indicated above suitable analytes include, but are not limited to
proteins, glycoproteins, antigen, antibodies, nucleic acids, sugars, carbohydrates, lectins,
and the like. However, larger, multimolecular, entities, such as cells, cell membranes and

1 5 other cellular constituents can also be detected and/or quantified by the methods of this
invention. Thus, for example, microorganisms {e.g. bacteria, fungi, algae, etc.) having
characteristic surface markers {e.g. receptors, lectins, etc.) can be detected and/or
quantified {e.g. in a biological sample from an animal, or plant). Similarly, cell types
{e.g. cells characteristic of a particular tissue) having characteristic markers {e.g. tumor

20 cells overexpressing IL-13 receptor {see, e.g., U.S. Patent 5,614,191)). Thus, cells

indicative of particular pathologies, particular states of differentiation (or lack thereof) or
particular tissue types can be detected and/or quantified.

Con jugation of the biological binding partner (ligand or antiligand) effector molecule
25 "chip" surface.

In one embodiment, the biological binding partner (ligand or antiligand) is
chemically conjugated to the underlying surface {e.g. the bio-electric interface.) Means of
chemically conjugating molecules are well known to those of skill {see, e.g., Chapter 4 in
Monoclonal Antibodies: Principles and Applications, Birch and Lennox, eds. John Wiley
30 & Sons, Inc. N.Y. (1995) which describes conjugation of antibodies to anticancer drugs,
labels including radio labels, enzymes, and the like).

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The procedure for attaching a binding partner (e.g. a protein, antibody,
glycoprotein, nucleic acid, lectin, sugar, carbohydrate, etc.) to a surface will vary
according to the chemical structure of the binding partner. Polypeptides typically contain
variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH2) groups,
5 which are available for reaction with a suitable functional on the surface or linker to
which they are to be bound.. Similarly, other biological molecules, e.g. nucleic acids,
sugars, carbohydrates, all contain a variety of functional groups (e.g. OH, NH2, COOH,
S, etc.) that are suitable points for linkage.

Alternatively, the targeting molecule and/or effector molecule may be

10 derivatized to expose or attach additional reactive functional groups. The derivatization
may involve attachment of any of a number of linker molecules such as those available
from Pierce Chemical Company, Rockford Illinois.

A "linker", as used herein, is a molecule that may be used to join the
biological binding partner (e.g. ligand or antiligand) to the underlying {e.g. apparatus or

1 5 device) surface. The linker is capable of forming covalent bonds to both the biological
binding partner and to the underlying surface. Suitable linkers are well known to those of
skill in the art and include, but are not limited to, straight or branched-chain carbon
linkers, heterocyclic carbon linkers, or peptide linkers.

A Afunctional linker having one functional group reactive with a group on

20 the surface, and another group reactive with the binding partner may be used to form the
desired conjugate. Alternatively, derivatization may involve chemical treatment of the
binding partner and/or the substrate. For example, a silica or glass substrate can be
silanized to create functional group. Similarly, a protein or glycoprotein, can be
derivatized, e.g., by glycol cleavage of a sugar moiety attached to the protein antibody

25 with periodate to generate free aldehyde groups. The free aldehyde groups on the

antibody or protein or glycoprotein may be reacted with free amine or hydrazine groups
on athe surface to bind the binding partner thereto (see U.S. Patent No. 4,671,958).
Procedures for generation of free sulfhydryl groups on polypeptide, such as antibodies or
antibody fragments, are also known (see U.S. Pat. No. 4,659,839).

30 Many procedures and linker molecules for attachment of various

biological molecules to various metal, glass, plastic etc., substrates are well known to
those of skill in the art. See, for example, European Patent Application No. 188,256; U.S.
Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and

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4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075. Methods of
conjugating antibodies, proteins, and glycoproteins abound in the immunotoxin literature
and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the
Magic Bullet," Thorpe et al, Monoclonal Antibodies in Clinical Medicine, Academic
5 Press, pp. 168-190 (1982), Waldmann, Science, 252: 1657 (1991), U.S. Patent Nos.
4,545,985 and 4,894,443.

Use of nucleic acid binding partners.

Where the binding partner is a nucleic acid (e.g. DNA, RNA, peptide

10 nucleic acid, etc.) specific binding is preferably achieved under "stringent" conditions, the
more stringent the conditions, the more specific the hybridization.

The selection of stringent conditions for any probe/target combination is
routinely accomplished by those of ordinary skill in the art. Moreover stringency can be
determined empirically by gradually increasing the stringency of the conditions (e.g.

15 increasing salt, raising temperature, etc.) until the desired level of specificity is obtained.

"Starting points" for stringent conditions are well known. For example,
desired nucleic acids will hybridize to complementary nucleic acid probes under the
hybridization and wash conditions of 50% formamide at 42°C. Other stringent
hybridization conditions may also be selected. Generally, stringent conditions are

20 selected to be about 5°C lower than the thermal melting point (T m ) for the specific

sequence at a defined ionic strength and pH. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe. Typically, stringent conditions will be those in which the salt
concentration is at least about 0.02 molar at pH 7 and the temperature is at least about

25 60°C. As other factors may significantly affect the stringency of hybridization, including,
among others, base composition and size of the complementary strands, the presence of
organic solvents and the extent of base mismatching, the combination of parameters is
more important than the absolute measure of any one. An extensive guide to
hybridization of nucleic acids is found in Ausubel et al, Current Protocols in Molecular

30 Biology current Protocols, a joint venture between Greene Publishing Associates, Inc and
John Wiley and Sons, Inc. (supplemented through 1998).

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Oligonucleotides for use as binding partners are chemically synthesized,
for example, according to the solid phase phosphoramidite triester method first described
by Beaucage, S.L. and Carruthers, M.H., 1981, Tetrahedron Lett., 22(20): 1859-1862
using an automated synthesizer, as described in Needham-VanDevanter, D.R., et al.,
5 1984, Nucleic Acids Res., 12:6159-6168. Purification of oligonucleotides is by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as described in
Pearson, J.D. and Regnier, F.E. (1983) J. Chrom. 255:137-149. The sequence of the
synthetic oligonucleotide can be verified using the chemical degradation method of
Maxam, A.M. and Gilbert, W. (1980) in Methods Enzymol. 65:499-560.

10 The bio-assay device will have a variety of uses, including for example,

screening large numbers of molecules for biological activity or screening biological
samples for the presence or absence concentration of a particular component or
components. To screen for biological activity, for example, the binding region is exposed
to one or more receptors, such as antibodies or whole cells. By detecting an interaction

15 between the binding region antiligand and the ligand, the presence and concentration can
be determined. A particular advantage of this technique is that no labels are needed to
detect this interaction. The inherent properties of the individual molecules are used to
detect their presence and amount, absence, or interaction with other molecules.

Other possible applications for the bio-assay device or chip include

20 diagnostics, in which various antibodies for particular receptors would be used to form
the binding region, and blood would be screened for immune deficiencies for example.
The bio-assay device is optionally fabricated such that it fits into a hypodermic needle
bore. Only a tiny blood sample would be necessary to detect a binding to a pre-applied
antiligand on the binding region. A diagnostic assay can be made to measure a whole

25 range of clinically relevant analytes, from pathogens such as viruses or bacteria, to
metabolic activities like glucose concentration or lipid levels, to the usual sets test for
liver enzymes, electrolytes, clotting factors, specific antibodies like ANA (used in
rheumatological disorders) and allergic response antibodies, arterial blood oxygenation,
drugs of abuse, and the like.

30 The bio-assay devices used in this capacity could be inexpensive

disposable chips, since they are easily fabricated and not limited to semiconductor
processing. For example, the chips are optionally fabricated on cheap materials like
plastic or glass substrates. The chips are then optionally placed in a device as described

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below and a signal propagated through the bio-assay device to detect the binding
interactions due to ligands in the blood. In fact, many different shapes and sizes of the
bio-assay devices could be fabricated containing various binding regions for the countless
biological and chemical applications for which detection without a label would be useful.
5 Unknown and uncharacterized proteins may be classified and/or identified

by detecting binding to structural motifs on the unknown protein. For example, proteins
in the same or similar class have structural homologies; that is to say, substructures such
as domains that recur within a given class of proteins. By fabricating a chip with multiple
addressable arrays, each of which has a antiligand for a specific substructure, an unknown

10 molecular species could be classified and/or identified as follows: The presence of
particular substructures is detected by the binding of each to its respective antiligand.
Each of these sub-structure binding events is then characterized by such qualities as
affinity, kinetics, and spectral response. Correlation is then made between the responses
of the unknown molecular species and data obtained from known proteins. In the case

15 that no exact fit is found, much of the structural details of the unknown compound can be
pieced together in much the same manner as NMR Spectroscopy does for organic
molecules.

In another embodiment, this technique may be used to develop gene chips
for the detection of nucleic acids. Gene chips are arrays of nucleic acids that are used for

20 the detection of complementary nucleic acids in a sample. The existence of the

complementary DNA, as measured by binding to distinct DNA molecules on the gene
chip, is the desired output. In the event that complementary binding does not occur,
partial hybridization can be detected and characterized by measuring such physical
quantities as affinity, melting point or other stringency conditions, and the direct spectral

25 response of the signal and correlation with previously measured data. In this manner, a
single antiligand in the form of a nucleic acid sequence can detect a whole range of
polymorphisms without the need for a separate sequence for each of the polymorphisms.
For example, a chip with just a few hundred different nucleic acid sequences could detect
tens of thousands of different polymorphisms.

30 Gene chips can be designed for the identification of drug targets, bacterial

identification, genotyping, and other diagnostics needs. The technique requires the
attachment of the requisite nucleic acids, typically as probes, onto a substrate and a
method to measure binding of complementary nucleic acids to that substrate. Ordinarily,

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the nucleic acids of the sample need to be labeled, most commonly with a fluorescent
probe. This technology eliminates the need for labeling the sample DNA and the
associated problems. Gene chips can be developed for specific needs in drug target
identification, molecular diagnostics, and detection and identification of biological
5 warfare agents. Other types of devices that could be fabricated and utilized are

immunoassay devices, drug discovery devices, and toxicity testing devices, analytical
devices, and the like.

The invention described herein can also be used for many aspects of new
drug development, from the initial screening process all the way though patient typing

1 0 and therapeutic monitoring. In the initial stages of drug discovery, the invention can be
used to facilitate target identification, validation, and high throughput screening (HTS).
Target receptors can be the antiligand on the bio-assay device, and by characterizing the
actions of known agonists, antagonists, or allosteric effectors, initial targets for the high
throughput screening procedure can be rapidly identified and validated. In the HTS

15 process, hundreds of thousands of compounds are tested to determine which of them can
bind to the target. The invention described herein can be miniaturized, so that highly
parallel screening platforms can be realized; platforms which are capable of screening
hundreds or thousands of compounds simultaneously, and at the same time determining
the effect of binding (e.g. agonist or antagonist), affinity, kinetics, etc. Additionally, such

20 miniature systems require very small amounts of compound, thus greatly saving costs in
purchasing said compounds from combinatorial libraries. The system of detection formed
through use of the bio-assay device provides a high throughput detection system because
detection optionally occurs in real time and many samples can be rapidly analyzed. The
response period is optionally monitored on a nanosecond time scale. As soon as the

25 molecules are bound to each other, detection occurs. More time is optionally required to
measure low concentrations or binding events between molecules with a low binding
affinity. The actual time is optionally limited by diffusion rates. Other than these
potential limitations, thousands of compounds are optionally run through the system very
quickly, for example, in an hour. For example, using chip fabrication technologies, a

30 10,000 channel device (using some of the emerging micro fluidics technologies) is
possible, and with small volumes and thus short diffusion times, and kinetic
measurements measuring only the beginning of the reaction, 10 million samples per hour
are optionally measured. With known concentrations, the binding affinity is optionally

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calculated from the kinetics alone and thus the device can be probed at a very fast time
scale and the affinity calculated and/or estimated from the slope of the kinetic curve.
References for kinetics and affinities can be found in any standard biochemistry or
chemistry text such as Mathews and van Holde, Biochemistry. Benjamin Cummings,
5 New York, 1990.

The invention may be easily extended into cell-based assays, since the
detection may not require sample purification and amplification. In these classes of
applications, cellular systems may be monitored for various changes either by detecting
external expressions or by lysing the cell to release the cytosolic constituents and detect

1 0 the presence of one or more analytes of interest.

The invention may also be adapted to "Laboratory-on-a-Chip"
applications. Because of the ease of miniaturization, very small chips with thousands or
tens of thousands of addressable bio-assay devices contained therein may be realized. The
detector may be realized as a sort of "logic gate" in which the presence of a particular

15 ligand or analyte has the effect of either turning on the gate or turning off the gate, as is
appropriate for a given application. Such a gate may be realized in any number of ways
which translate the binding event into an electromagnetic signal which can be assigned to
one of two possible states corresponding to off and on, 1 or 0, and the like. The two states
could be different frequencies of a resonant cavity or waveguide corresponding to bound

20 and unbound, or amplitude changes in a transmission line or waveguide which correspond
to bound and unbound, or changes in the band-pass of a particular circuit, or the like.

While the above is a complete description of possible embodiments of the
invention, various alternatives, modifications, and equivalents may be used. For instance
a person skilled in the art will appreciate that the signal path of foregoing bio-assay

25 device is not limited to a transmission line. Other transmission mediums, such as

conductive or dielectric waveguides may alternatively be used. Further, all publications
and patent documents recited in this application are incorporated by reference in their
entirety for all purposes to the same extent as if each individual publication and patent .
document was so individually denoted. The above description should be view as only

30 exemplary embodiments of the invention, the boundaries of which are appropriately
defined by the metes and bounds of the following claims.

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WHAT IS CLAIMED IS:

PCT/US99/17508

1 1. A method for detecting one or more properties associated with a

2 molecular binding region, the method comprising the steps of:

3 providing a molecular binding region coupled along the surface of a signal

4 path; and

5 propagating a test signal along said signal path, wherein said test signal

6 couples to said molecular binding region, and in response, exhibits a signal response.

1 2. The method of claim 1, further comprising the step of measuring

2 said signal response.

1 3. The method of claim 1 , wherein said surface of said signal path is

2 derivatized.

1 4. The method of claim 1 , wherein said molecular binding region

2 comprises an antiligand bound to a ligand.

1 5, The method of claim 1, further comprising the step of adding an

2 analyte to said molecular binding region, wherein said added analyte interacts with said

3 molecular binding region.

1 6. The method of claim 1 , wherein said step of adding occurs prior to

2 said step of propagating said test signal.

1 7. The method of claim 6, wherein the analyte is in a solution.

1 8. The method of claim 7, wherein the solution comprises a sample of

2 body fluid.

1 9. The method of claim 1, wherein the signal path comprises a second

2 electromagnetically coupled original path. .

1
2

1 0. A method for detecting one or more molecular binding events
between a ligand and an antiligand, the method comprising the steps of:

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3 exposing a portion of a signal path to a first solution containing a first

4 ligand, said exposed signal path portion comprising a first molecular binding region,

5 wherein said exposed signal path portion comprises a continuous transmission line; and

6 propagating a first test signal along said signal path, wherein said test

7 signal couples to said molecular binding region and exhibits a first signal response

8 indicating detection of said binding event between said first ligand and said antiligand.

1 11. The method of claim 1 0, further comprising the steps of:

2 exposing said portion of a signal path to a second solution containing a

3 second ligand; and

4 propagating a second test signal along said signal path, wherein said test

5 signal couples to said molecular binding region and exhibits a second signal response

6 indicating detection of said binding event between said second ligand and said antiligand.

1 12. The method of claim 10, wherein the antiligand is an antibody.

1 13. A method for detecting one or more properties associated with a

2 molecular binding region, the method comprising the steps of:

3 providing a molecular binding region coupled along the surface of a signal

4 path; and

5 propagating a test signal along said signal path, wherein the tangent of said

6 surface of said signal path is non-orthogonal to the direction of signal propagation of said

7 test signal,

8 wherein said test signal couples to said molecular binding region, and in

9 response, exhibits a signal response.

1 1 4. An apparatus for detecting one or more properties associated with a

2 molecular binding region, the apparatus comprising:

3 a signal path having an input signal port, an output signal port, and a

4 continuous conductive region therebetween; and

5 said molecular binding region coupled to said signal path.

1 15. The apparatus of claim 14, wherein said input signal port and said

2 output signal port comprises the same physical port.

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1 16. The apparatus of claim 14, wherein said input signal port and said

2 output signal port comprise two physically separated ports.

1 17. The apparatus of claim 1 4, wherein said molecular binding region

2 comprises a ligand/antiligand complex.

1 1 8. The apparatus of claim 14, wherein said molecular binding region

2 comprises a derivatized conductive layer.

1 19. The apparatus of claim 1 4, further comprising a retaining structure

2 for retaining a solution along said signal path.

1 20. The apparatus of claim 14, wherein said test signal propagates at a

2 frequency of greater than 1 MHz.

1 21. The apparatus of claim 1 4, wherein signal path comprises a

2 transmission line structure.

1 22. The apparatus of claim 14, wherein the signal path comprises a

2 resonant cavity.

1 23. A method for detecting one or more molecular binding events

2 between a Iigand and an antiligand, the method comprising the steps of:

3 applying a first solution and a first ligand over a portion of a signal path,

4 wherein a first molecular binding region comprising said antiligand has previously

5 formed along the surface of said portion of said signal path, said molecular binding region

6 being positioned more proximal to said signal path then said solution; and

7 propagating a test signal along said signal path, said signal path

8 comprising a path which is continuous along said surface, wherein said test signal couples

9 to said molecular binding region comprising a ligand/antiligand complex and exhibits a
1 0 first signal response.

24. A method for detecting one or more molecular binding events
between a ligand and an antiligand, the method comprising the steps of:

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3 applying a solution and a ligand over a signal path, wherein a molecular

4 binding region comprising said antiligand has previously formed along at least the surface

5 of at least a portion said signal path; and

6 propagating a test signal along said signal path, said signal path

7 comprising a non-orthogonal path relative to said surface, wherein said test signal couples

8 to said molecular binding region comprising a ligand/antiligand complex and exhibits a

9 first signal response.

1 25. The method of claim 24, further comprising the steps

2 storing said signal first signal response;

3 applying a second solution containing a ligand or antiligand over said

4 portion of said signal path;

5 forming a second molecular binding region along said signal path, said

6 second molecular region comprising said ligand and said antiligand,

7 propagating a second test signal along said conductive surface, wherein

8 said second test signal couples to said molecular binding region and in response exhibits a

9 second signal response, said second signal response being uncorrelated with said first
10 signal response.

1 26. The method of claim 24, further comprising the steps:

2 propagating a second test signal along said signal path to obtain a second

3 signal response;

4 comparing said first and second signal responses; and

5 determining said dielectric properties of said solution have changed if said

6 second response does not correlate with said first response within a predefined range.

1 27. The method of claim 26, further comprising the steps of:

2 correlating said first signal response to a first known dielectric property;

3 correlating said second signal response to a second known dielectric

4 property; and

5 removing the quantity of said first dielectric property from the quantity of

6 said second known dielectric property.

1 28. A method for determining the classification of an unknown ligand,

2 the method comprising the steps of:

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3 providing a signal path coupled to a first molecular binding region, said

4 molecular binding region comprising N respective antiligands for binding to N respective

5 ligand sub-structures;

6 applying a solution containing a plurality of unknown ligands over said

7 molecular binding region.

8 forming, in response, a second molecular binding region along said signal

9 path, said second molecular binding region comprising said N antiligands;

1 0 propagating N test signals to said N antiligands;

1 1 providing N known signal responses, said N known responses defining a

12 known classification of ligands;

13 wherein each of said N test signals couples to said N antiligands, and in

14 response exhibits N respective measured responses indicative of the presence of each of

1 5 said N sub-structures;

1 6 wherein if a predetermined number of said N known signal responses

1 7 correlates within a predefined range with said N measured responses, determining said

1 8 unknown ligand is within said known classification.

1 29. A method for identifying an unknown molecular binding event, the

2 method comprising the steps of:

3 providing a signal path;

4 applying a first solution containing a first ligand over said signal path;

5 forming, in response, a first molecular binding region along said signal

6 path, said first molecular region comprising said first ligand, wherein said first molecular

7 binding region is coupled along the surface of the signal path;

8 propagating a first test signal along said signal path, wherein said first test

9 signal couples to said molecular binding region and in response exhibits a first signal

10 response;

1 1 providing a known signal response corresponding to a known molecular

12 binding event;

13 comparing said first signal response with said known signal response,

14 wherein if said first signal response correlates to said known signal response within a

15 predefined range, said unknown molecular binding event comprises said known

16 molecular binding event.

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1 30. A method for quantitating an unknown concentration of ligands in

2 a solution comprising the steps of:

3 providing a signal path coupled to a first molecular binding region, said

4 molecular binding region comprising at least one antiligand;

5 applying a solution having a known concentration of ligands over said

6 molecular binding region to obtain a first signal response from a propagated test signal;

7 repeating said applying step in one or more different known

8 concentrations;

9 correlating the signals with the known concentrations;

10 measuring a second signal response to a propagated test signal; and

1 1 correlating the second signal response to said algorithm.

1 3 1 . An apparatus for detecting the presence of a ligand or antiligand,

2 comprising:

3 a signal path comprising a continuous conductive region;

4 a molecular binding region coupled to at least a portion of said continuous

5 conductive region, said molecular binding region comprising said ligand or antiligand;

6 and

7 a solution coupled to said molecular binding region.

1 32. A bio-electrical interface for detecting the presence of a ligand in a

2 solution, comprising:

3 a signal path comprising a continuous conductive region;

4 a solution for providing said ligand; and

5 a molecular binding region coupled along said signal path and said

6 solution, said molecular binding region comprising said ligand.

1 33. The bio-electrical interface of claim 32, further comprising:

2 a ground plane; and

3 a dielectric layer coupled between said ground plane and said solution.

1 34. The bio-electrical interface of claim 32, wherein said molecular

2 binding region operates as a shunt circuit coupled between said transmission path and

3 said solution.

WO 00/45160

PCT/US99/17508

1 35. The bio-electrical interface of claim 32, wherein said solution

2 operates a shunt circuit coupled between said shunt MBR circuit and said ground plane.

1 36. The bio-electrical interface of claim 35, wherein said molecular

2 binding region comprises the electrical characteristics of a series R-L circuit coupled

3 along said signal path.

1 37. The bio-electrical interface of claim 36, wherein said solution

2 comprises the electrical characteristics of a series R-L circuit coupled along said signal

3 path.

1 38. The bio-electrical interface of claim 32, further comprising:

2 a ground plane; and

3 a dielectric layer coupled between said signal path and said ground

4 plane.

1 39. The bio-electrical interface of claim 3 8, wherein said molecular

2 binding region operates as a shunt circuit coupled to said signal path.

1 40. The bio-electrical interface of claim 39, wherein said solution

2 comprises the electrical characteristics of a parallel L-R-C circuit coupled to said

3 molecular binding region.

1 4 1 . The bio-electrical of claim 32, wherein the molecular binding

2 region is a proximal to a ground plane.

1 42. The bio-electrical of claim 32, wherein the molecular binding

2 region is proximal to both signal and ground planes.

1 43. The bio-electrical interface of claim 32, wherein the molecular

2 binding region is proximal to a portion of a wave guide.

1 44. The bio-electrical interface of claim 32, wherein the molecular

2 binding region is proximal to a portion of a micro strip.

1 45. The bio-electrical interface of claim 32, wherein the molecular

2 binding is proximal to and incorporated in a portion of a resonant cavity.

WO 00/45160 PCT/US99/17508

1 46. A bio-electrical interface of claim 32, wherein the signal path

2 comprises an input original path and an output signal path.

1 47. An apparatus for detecting the presence of a ligand using a test

2 signal, the apparatus comprising:

3 a signal path comprising a continuous conductive region and having a first

4 port and a second port for communicating said test signal therebetween;

5 a molecular binding region coupled to said signal path, said molecular

6 binding region comprising said ligand; and

7 a solution coupled to said molecular binding region for transporting said

8 ligand to said molecular binding region.

1 .48. The apparatus of claim 47, wherein the solution is a body fluid.

1 49. The apparatus of claim 48, wherein the body fluid is blood.

1 50. A system for detecting a molecular binding event, comprising:

2 a signal source for launching a test signal;

3 a bio-assay device coupled to said signal source, comprising:

4 a signal path comprising a continuous conductive region;

5 a solution contain a ligand for producing said molecular binding

6 event; and

7 a first molecular binding region comprising said ligand; and

8 a signal detector coupled to said signal path,

9 wherein said test signal propagates along said signal path and couples to
10 said molecular binding region comprising said ligand, and in response exhibits a signal

- 1 1 response, said signal response indicating the presence of said molecular binding event.

1 51. The test system of claim 50, wherein said test signal comprises a

2 frequency-varying signal and wherein said signal response comprises a transmission loss

3 S21 frequency response of said test signal.

1
2
3

52. The test system of claim 50, wherein said test signal comprises a
frequency- varying signal and wherein said signal response comprises a return loss Si 1
frequency response of said test signal.

WO 00/45160 PCT/US99/1 7508

1 53. The test system of claim 5 1 or 52, wherein said test signal

2 comprises a frequency, varying signal which is a resonant response.

1 54. The test system of claim 51 or 52, wherein said test signal

2 comprises a frequency, varying signal which is a non-resonant response.

1 55 The test system of claim 51 or 52, wherein said test signal

2 comprises a pure frequency or a frequency varying signals and wherein said signal

3 response comprises a shift in one or more of said frequencies.

1 56. The test system of claim 50, wherein said test signal comprises a

2 time domain waveform and said signal response comprises a transmitted time domain

3 response.

1 57. The test system of claim 50, wherein said test signal comprises a

2 time domain waveform and said signal response comprises a reflected time domain

3 waveform.

1 58. The test system of claim 50, wherein said test signal comprises a

2 time domain waveform of varying pulse intervals and said signal response comprises a

3 reflected time domain waveform.

1 59. In a computer-controlled molecular binding event detection system

2 for use with a bio-assay device having a molecular binding region coupled along a signal,

3 a computer program product for detecting one or more properties associated with the

4 molecular binding region, the computer program product comprising:

5 code that directs said processor to instruct said system to propagate

6 a test signal along said signal path, wherein said test signal couples to said molecular

7 binding region, and in response, exhibits a signal response; and

8 a computer readable storage medium for storing said code.

1 60. The computer program product of claim 59 further comprising

2 code that directs said processor to instruct said system to measure said signal response.

1 61. In a computer-controlled molecular binding event detection system

2 for use with a bio-assay device having a molecular binding region coupled along a signal,

WO 00/45160 g7 PCT/US99/17508

3 a computer program product for detecting one or more molecular binding events between

4 a ligand and antiligand, the computer program product comprising:

5 code that directs said processor to instruct said system to apply a

6 first solution to a portion of said signal path; and

7 code that directs said processor to instruct said system to propagate

8 a first test signal along said signal path, wherein said test signal couples to said molecular

9 binding region and exhibits a first signal response indicating detection of said binding

10 event between said first ligand and said antiligand; and

11 a computer readable storage medium for storing said code.

1 62 . The computer program product of claim 6 1 , further comprising:

2 code that directs said processor to instruct said system to expose said

3 portion of a signal path to a second solution containing a second ligand; and

4 code that directs said processor to instruct said system to propagate a

5 second test signal along said signal path, wherein said test signal couples to said

6 molecular binding region and exhibits a second signal response indicating detection of

7 said binding event between said second ligand and said antiligand.

1 63. In a computer-controlled molecular binding event detection system

2 for use with a bio-assay device having a signal path comprising a continuous conductive

3 region coupled to molecular binding region comprising N antiligands for binding to N

4 respective ligand substructures, a computer program product for determining the

5 classification of an unknown ligand contained in a solution, the computer program

6 product comprising: .

7 code that directs said processor to instruct said system to apply said

8 solution containing a plurality of said unknown ligands over said molecular binding

9 region, wherein a second molecular binding region forms along said signal path, said

10 second molecular binding region comprising said N anti ligands;

1 1 code that directs said processor to instruct said system to propagate N test

12 signals to said N antiligands;

13 code that directs said processor to instruct said system to provide N known

14 signal responses, said N known responses defining a known classification of ligands,

15 wherein each of said N test signals couples to said N antiligands, and in response exhibits

WO 00/45160 88 PCT/US99/17508

1 6 N respective measured responses indicative of the presence of each of said N sub-

17 structures;

18 code that directs said processor to instruct said system to determine if a

1 9 predetermined number of said N known signal responses correlates within a predefined

20 range with said N measured responses; and

2 1 a computer readable storage medium for storing said code

1 64. In a computer-controlled molecular binding event detection system

2 for use with a bio-assay device having a molecular binding region coupled along a signal,

3 a computer program product for quantitating an unknown concentration of ligands in a

4 solution, the computer program product comprising:

5 code that directs said processor to instruct said system to apply a solution

6 having a known concentration of ligands over said molecular binding region to obtain a

7 first signal response from a propagated test signal;

8 code that directs said processor to instruct said system to repeat said

9 applying step in one or more different known concentrations;

I o code that directs said processor to instruct said system to correlate the

I I signals with the known concentrations;

12 code that directs said processor to instruct said system to measure a second

1 3 signal response to a propagated test signal;

14 code that directs said processor to instruct said system to correlate the

1 5 second signal response to said algorithm; and

16 a computer readable storage medium for storing said code.

WO 00/45160

PCT/US99/17508

FIG. 1B

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2/28

176.

176c

FIG. 1C

LIGAND
ANTILIGAND
LINKER

MATRIX

LINKER
INSULATING
LAYER
LINKER

SOLUTION

180

CONDUCTIVE
LAYER

FIG. 1D

WO 00/45160

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WO 00/45160

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PCT/US99/17508

153

FIG. 1H

WO 00/45160

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5/28

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6/28

WO 00/45160

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PCT/US99/17508

SOLUTION
350

MOLECULAR
BINDING S
LAYER

340

CONDUCTOR
SUBSTRATE-

330

•>•<•; METAL: V.

^fe — — 1

W ; / ■ //

' //// ".
/ / / / /
'/////

+ l

320

FIG. 3

WO 00/45160

PCT/US99/17508

8/28

03
CO
CM

OS
C\J
C\J

CO
UJ

DC
LU

ERI

T.L

LU_J

ERI

03
CO
CM

CM
CM

CO
UJ

DC
UJ
CO

-Q

3 O

co o

COQj
Q

03
CM

ERI

T.L

LU_J

ERI

WO 00/45160

9/28

PCT/US99/17508

WO 00/45160

10/28

PCT/US99/17508

CO
CNJ
LO

ERI

T.L

o
in

-Q

—J

—I

CO
CM
LO

ERI

T.L

WO 00/45160

PCT/US99/17508

WO 00/45160

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12/28

-510

530

550

FIG. 5A

r 510

550

FIG. 5B

520-^ ^-510

550

515

FIG. 5C

520

530

-530

550^ 7 ^ 515

510-- 7

F/G. 5D

^-510

550 ^ 520-^ jL^ 515 550

■ N ' ' ' 1 -530

F/G. 5E

51 0-^ 5]5 ^550

550"^ 5 2o

-530

F/G. 5F

WO 00/45160

PCT/US99/17508

13/28

570

FIG. 5G

PROVIDE SIGNAL PATH

PROVIDE SOLUTION CONTAINING THE
SUBJECT MOLECULE OR STRUCTURE

602

604

FORM MBL BETWEEN
SIGNAL PATH AND SOLUTION

PROPAGATE TEST SIGNAL
ALONG SIGNAL PATH

TEST SIGNAL COUPLES TO MBL
AND DEVELOPS SIGNAL RESPONSE

RECOVER SIGNAL RESPONSE

SIGNAL RESPONSE ENABLES
DETECTION OF SUBJECT MOLECULE
OR STRUCTURE

-606

-608

-610

-612

-614

FIG. 6A

WO 00/45160

14/28

PCT/US99/17508

PROVIDE BINDING DETECTED AND
SIGNAL RESPONSE MEASURED

PRIMARY BINDING SIGNAL
RESPONSE IS STORED

SECOND SAMPLE IS ADDED
TO BIO-ASSAY CHIP

SECONDARY BINDING
RESPONSE MEASURED

COMPARE PRIMARY &
SECONDARY RESPONSES

-620

-622

-624

626

628

SECONDARY BINDING
EVENT HAS OCCURRED

J\YES

630

SECONDARY BINDING

EVENT HAS NOT OCCURRED

FIG. 6B

WO 00/45160

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15/28

MEASURE & RECORD FIRST SIGNAL
RESPONSE OF SOLUTION

MEASURE & RECORD SECOND SIGNAL
RESPONSE OF SOLUTION

COMPARE FIRST AND
SECOND SIGNAL RESPONSES

645

-641

-642

-643

YES

QUALITY HAS
CHANGED

644

QUALITY HAS
NOT CHANGED

CORRELATE SECOND
RESPONSE TO KNOWN
RESPONSES

DETERMINE QUALITY
CHANGED & MAGNITUDE

-646

•647

FIG. 6C

CHARACTERIZE N MOLECULAR
STRUCTURES & STORE RESPONSES

MEASURE UNKNOWN SAMPLE

COMPARE UNKNOWN RESPONSES
TO KNOWN RESPONSES

CLOSEST CORRELATED KNOWN
RESPONSE IDENTIFIES UNKNOWN
MOLECULAR STRUCTURE

-652

654

-656

-658

FIG. 6D

WO 00/45160

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16/28

PROVIDE BIO-ASSAY CHIP HAVING
MULTIPLE ADDRESSABLE ARRAYS

DETECT PRESENCE OF EACH SUB-
STRUCTURE AT EACH ARRAY MARKER

CHARACTERIZE EACH BINDING
EVENT FOR IDENTIFICATION

CORRELATE UNKNOWN RESPONSE OF
EACH SUBSTRUCTURE WITH
KNOWN RESPONSES

660

662

-664

666

YES

UNKNOWN MOLECULE IS

WITHIN CLASS OF
MOLECULES EXHIBITING
CORRELATION THEREWITH

UNKNOWN MOLECULE
CORRESPONDS TO
KNOWN RESPONSES

FIG. 6E

WO 00/45160

PCT/US99/17508

17/28

679

IS"
DEVICE
PRECALIBRATED,

CHOOSE ANTILIGAND WITH
APPROPRIATE AFFINITY FOR
ANALYTE BEING MEASURED
(LINEAR OPERATING RANGE)

675a-

SPECTRAL ANALYSIS:

DETERMINE
FREQUENCIES WHERE
ANALYTE BINDING HAS
MAXIMAL EFFECT

675b

SPECTRAL ANALYSIS:
DETERMINE REGIONS
WHERE NON-SPECIFIC
BINDING HAS
MAXIMAL EFFECT

ATTACH ANTILIGAND TO
BIO-ELECTRIC DEVICE

PLACE DEVICE IN
DIAGNOSTIC DEVICE
(E.G. NETWORK ANALYZER)

-670

-671

-673

YES

-675c

'NEED TO
CHARACTERIZE
"RESPONSE FOR MAXIMAL.
SPECIFICITY?

674

676a

SPECTRAL ANALYSIS:
DETERMINE UNIQUE
RESPONSE DUE TO
ANALYTE BINDING

PERFORM CALIBRATION BY
MEASURING RESPONSE

AGAINST SAMPLE OF
KNOWN CONCENTRATION

676c

CHOOSE
SAMPLE WITH
DIFFERENT
CONCENTRATION

677.

SET UP EXTRAPOLATION
ALGORITHM WITH
CALIBRATION DATA

678a

678

APPLY UNKNOWN SAMPLE

COMPARE RESPONSE
WITH EXTRAPOLATION
ALGORITHM
AND DETERMINE
CONCENTRATION

FIG. 6F

WO 00/45160

18/28

PCT/US99/17508

GENERATE QUANTITATIVE REPONSE
CURVE FOR ANTILIGAND BINDING TO
PREDETERMINE TITRATION V

GENERATE QUANTITATIVE REPONSE
FOR LIGAND BINDING TO ANTILIGAND
TO PRE-DETERMINE TITRATION CURVE

CALCULATE RESPONSE
SCALE FACTOR "A"

PROPAGATE SIGNAL THROUGH
UNCALIBRATED ASSAY AT POINT-OF-
USE AND DETERMINE AMOUNT "x" OF

ANTILIGAND BOUND TO ASSAY

CALCULATE SCALE FACTOR "y"
FROM "A" AND "x"

APPLY LIGAND TO ASSAY
AND MEASURE RESPONSE

DETERMINE LIGAND CONCENTRATION
BY MULTIPLYING BOTH RESPONSE AND
PRE-DETERMINED TITRATION CURVE BY
SCALE FACTOR "y" AND COMPARING

-680

-682

-684

-686

688

-689

-690

FIG. 6G

WO 00/45160

PCT/US99/17508

19/28

710

n nmn n=y=n mf=pi an'n \

, ggga \ \ ^734

F/G. 7/4

724

I/O

CONTROLLER

^-740

•726

-728

SYSTEM
MEMORY

CENTRAL
PROCESSOR

710

730

SPEAKER

-740

DISPLAY
ADAPTER

714

MONITOR

REMOVABLE
DISK

734

KEYBOARD

-736

FIXED
DISK

^-738

NETWORK
INTERFACE

FIG. 7B

WO 00/45160

PCT/US99/I7508

20/28

810

860

810

852

858

SOURCE

BIO-ASSAY

SOURCE

DEVICE

DETECTOR

880

850

870

DETECTOR

890

FIG. 8A

fstart

'res

FREQUENCY

REF

FIG. 8B

SLOPE

AMPLITUDE
VARIATION
REF

fstart

fstop

FIG. 8C

WO 00/45160

PCT/US99/17508

21/28

920

910

930

922a

912

922

1002

SIGNAL
SOURCE

SIGNAL
DETECTOR

1004

FIG. 9

1060

S
_TL

1000

1070

FIG. 10

1002

028

TEST

SIGNAL

CIRCUIT

SOURCE

1020

SIGNAL

DETECTOR

1004

1122

1128

1100

/ / r

1162 1164 |

SIGNAL
DETECTOR

1120

1104

/ (ABSORBED
1170 P ° WER)

1102

TEST

SIGNAL

CIRCUIT

SOURCE

SIGNAL
DETECTOR

"V"

1104

FIG. 11

WO 00/45160

PCT/US99/17508

22/28

+0.5dB

OdB

-0.5dB

45MHz

FIG. 12A

1GHz

+0.8dB

OdB

-0.2dB

45MHz

1GHz

FIG. 12B

WO 00/45160

PCT/US99/17508

23/28

+2.5dBT

OdB

-2.5dB

i .

- !

!
!
1

-r*

i ;

| ;

! !

1GHz

FIG. 12C

10GHz

+2.5dB

OdB

-2.5dB
1GHz

10GHz

FIG. 12D

WO 00/45160

PCT/US99/17508

24/28

i i

1 i

r i i

!
I

BOUND

UNBOUND

FIG. 12E

+2.5dB

OdB

-2.5dB

— ^

- — s

1254

•1252

1GHz

20GHz

FIG. 12F

WO 00/45160

PCT/US99/17508

25/28

+2.5dB

OdB

-2.5dB

-i-

1GHz

FIG. 12G

1264

1262

20GHz

400-
350-
300-
250-

£ 200-

CL
CO
HI

150-
100-
50-
0-
-50-

i — r
2

i — r

PERCENT
BOUND

100%

50%

r " E r"T =F
8 10

1 1 r

12 14

CONCENTRATION (mg/dl)

FIG. 12H

WO 00/45160 PCT/US99/17508

26/28

+2.5dB

OdB

-2.5dB

r
\

3GHz

FIG. 121

4.6GHz

+1.5dB

OdB

-1 .5dB

1GHz

10GHz

FIG. 12J

WO 00/45160

PCT/US99/17S08

28/28

4 5 6 7
SAMPLE NUMBER

FIG. 12M

+2.5dB

OdB

-2.5dBl

1GHz

V—

— i

20GHz

FIG. 12N

INTERNATIONAL SEARCH REPORT

Best Available Copy

Interna .1 Application No _ _ ~ «

T?CT/US 99/17508

A. CUSSIRCATION OP SUMKCT MAITIiK

G01N27/327,G01N33/543,C12Ql/00,C12Ql/68,G06F19/00

According to International Patent Oacnfication (IPC) or to hoch natfonal clasificaUon and IPC 7

ii. r-'ii;u>ssi:Aiictii;i>

Minimum documcnlauon searched (duuificaUon xyncm followed by daraQcauon symboli)

G01N,C12Q,G06F

Documentation searched other than minimum documentation to the extern Out such documents arc included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category ' Citation of document, with indication, where approoriate, of the relevant passages

Relevant to claim No.

X, E

WO 99/39190 A

(SIGNATURE BIOSCIENCE INC.)
05 August 1999,
abstract, claims, fig..

US 4822566 A

(NEWMAN, A.L. ) 18 April 1989,
the whole document.

1-64

WO 97/41425

(pence;

1997,
pages 3-

INC) 06 November
5, fig..

WO 96/36871 A

(AUSTRALIAN MEMBRANE AND

10,

,14,

,24,

-32,

,50

10,

,14,

,24,

-32,

,50

14,

,32,

X I FurLhcr document! are listed in the continuation of box C.

Patent family members are listed in annex.

Special categories of cited documents :

* A" document defining the ceneral sUte of ihe art which it not
considered to be of particular relevance

'U' earlier document but published on Or after- the international
filing dale

'L # document which may throw doubts on priority daimfs) or '
which is cited to crubJiih the publication date of another
citation or other xpeciai reason (as specified)
*0" document referring to an oral disclosure, use, exhibition or
other means

P* document published prior to the international filing dale but
later than the priority date daimed *

T later document published after the international filing date
or priority date and not in conflict with the application but
dtcd to understand the principle or theory underlying the
invention

"X" document of particular relevance; the daimed invention
cannot be considered novd or cannot be considered to
involve an inventive step when the document it taken alone

*Y* document of particular relevance; the daimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combtnauon being obvious to a person skilled
in the art.

'&.' document member of the same patent family

Date of the actual -completion of (he international search

22 November 1999

Name and mailing address of (he ISA

European Patent Office, P.O. 581 S Paicmlaan 2
NL - 2280 HV Rijswijk
Td.( + 31-70) 340-2040, Tx. 31 651 cpo rd.
Fax: 31-70) 340-3016 • •

Date of mailing of the international search report

21.12.99

Authorized offico-

SCHNASS e.h.

Fan. PCT7ISA/JI0 (■««< , Kca) (/...., {m \

INTERNATIONAL SEARCH REPORT

i I

iMcm il Application No •

PCT/US 99/1750E

C/ConlinuaUon) DOCUMENTS CONSIDERED TO DE RELEVANT

Category *

Citation of document, with indication, where appropriate, of the relevant passages R

c levant to claim No.

BIOTECHNOLOGY RESEARCH
INSTITUTE) 21 November 199b,
pages 1-6, examples.

Form PCT/KA/3ia (oonilnunioo of leoond iheet) (July UT2)

Best Available Copy

zub internationalen Recherchen-
bericht fiber die Internationale
Patentafweldung Kr.

to the International &arch
Report to the International Patent

ANNEXE

Application
PCT/US 99/17508 5AE 244045

nat*™.-.». .
intarsaitaul n

This Annex lists the patent teiil ly
oeabers relating to the patent docuaents
t cited in the above-cent iotm 'inter-,
national search report. The Office is
in no way liable far these particulars

In dieses Anhara sind die Mitglieder
der PatenHanihen der « riwger
nannten international en Recherchenbericht
anqe^hrten Paten tdokurente angegeben.

ot wtornauon. de l'Office. ,

U nresente annexe indique Jes
raiafres de la f am lie de brevets
relatlfs aux docuaents oe brevets cites
dans le rapport de recherche inter;
national visee cHJeeag. }«J««9K , 1I
its foumis sont donnes a titre indica-

]a Recherchenbericht
anoefuhrtes Paten tdokument
Tatent docuaent cited
in search report
Document de brevet cite
dans le rapport de recherche

Datua der
Verfiffentlichung
Publication
date
Date de
publication

KHgUed(er) der
PaTantfaailie
Patent faaily

Renbre(s) de la
faille de brevets

Datua der
VerfiHentlichung
Publication

date
Date de
publication

UJ0_A1_
US A

9939 190_
~4B225&6

05-08-^999
Tb-04-19B9

keine? - none

r ien

CA Al
EP Al
JP T2
WO Al

1259374
245477
63501446
8703095

12-09
19-11

02-06
21-05

989

,987
•19BB
•1987

WO Al

9741425

06-11-1997

AU Al
CA AA
LIB A
AU Al
CA AA
EP Al
WO Al

25639/97
2251674
5955379

25638/97
2252474
895592
9741424

19-11-
06-11-

21-09-
19-11-

06-11-
10-02-

06-11-

•1997
-1997
•1999
-1997

-1999
1997

THIS PAGE BLANK (uspto)

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